Compounds, Compositions, and Methods for Protecting Brain Health in Neurodegenerative Disorders

ABSTRACT

Aspects of the invention relate to compounds, extracts and compositions thereof, and methods of using of the same, to treat neurodegenerative disorders and/or improve brain health. In certain embodiments, said compounds are pomegranate flavonoids.

RELATED APPLICATION

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 61/228,374, filed Jul. 24, 2009; thecontents of which are hereby incorporated by reference.

BACKGROUND

Neurodegenerative disorders and brain health are among the majoremerging public health challenges facing our aging society. Alzheimer'sdisease (AD) is by far the most prevalent of the neurodegenerativedisorders. Several aging pathologies, such as Parkinson's disease,Huntington's disease and ALS, are known to share some pathophysiologywith Alzheimer's disease, including aberrant protein folding andoxidative stress. Sayre, L. M., G. Perry, et al. (2008). “Oxidativestress and neurotoxicity.” Chem Res Toxicol 21(1): 172-88.

In addition to selective neuronal degeneration, AD is characterizedpathologically by the presence of two hallmark lesions in the brain:extracellular senile plaques (SP) and intraneuronal neurofibrillarytangles (NFT). SP contain amyloid-β (Aβ) peptides, primarily Aβ(1-42);whereas, NFT are composed mainly of the microtubule-associated proteinTau in the form of paired helical filaments. The pathophysiology of ADis also characterized by increased production of soluble peptides ofAmyloid Beta—peptides Aβ(1-40) and Aβ(1-42). Some recent findingssuggest that neuronal toxicity and compromised synaptic transmission maybe due to increased production of soluble oligomers of Aβ.

A need exists for compounds and compositions that treatneurodegenerative disorders and/or improve brain health. Ideally, suchcompounds would have good pharmaceutical properties, such as solubility,bioavailability and/or few side effects.

SUMMARY

Certain aspects of the invention relate compounds, extracts andcompositions thereof, and methods of using them to treatneurodegenerative disorders and/or improve brain health. In certainembodiments, said compounds are pomegranate flavonoids. In certainembodiments, said compounds, extracts, and/or compositions may be usedfor the treatment, management or prevention of a disease or conditionassociated with the damage induced by fibril formation, or the increasedrisk of fibril formation.

In one aspect, the invention relates to a composition comprising acompound or extract of the invention, such as a pharmaceuticalcomposition, a nutraceutical formulation, a medical food (also known asdietary food for special medical purpose), a functional food, a foodadditive, or a dietary supplement (also known as phytomedical product),comprising one or more anti-aggregation compounds of the invention. Thecompositions may also contain an additional therapeutic agent, or may beadministered in combination with another therapeutic compound. Otheraspects of the invention relate to packaged products containing theabove-mentioned compositions and a label and/or instructions for use inpreventing aggregation in a patient at risk, for the treatment of adisease or condition associated with damage to the brain associated withtypical aging diseases and/or for preventing and/or managing associatedcell death.

Additional aspects, embodiments, and advantages of the invention arediscussed below in detail. Moreover, the foregoing information and thefollowing detailed description are merely illustrative examples ofvarious aspects and embodiments of the invention, and are intended toprovide an overview or framework for understanding the nature andcharacter of the claimed aspects and embodiments. The accompanyingdrawings are included to further understanding of the various aspectsand embodiments, and are incorporated in and constitute a part of thisspecification. The drawings, together with the remainder of thespecification, serve to explain principles and operations of thedescribed and claimed aspects and embodiments. Various aspects of atleast one embodiment are discussed below with reference to theaccompanying figures. The figures are provided for the purposes ofillustration and explanation and are not intended as a definition of thelimits of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts selected compounds of the invention.

FIG. 2 depicts the ¹H NMR spectrum of Punicalagin.

FIG. 3 depicts the mass spectrum of Punicalagin.

FIG. 4 depicts the ¹H NMR spectrum of Punicalin.

FIG. 5 depicts the mass spectrum of Punicalin.

FIG. 6 depicts the ¹H NMR spectrum of Tellimagrandin.

FIG. 7 depicts the mass spectrum of Tellimagrandin.

FIG. 8 depicts the ¹H NMR spectrum of Compound A.

FIG. 9 depicts the mass spectrum of Compound A.

FIG. 10 depicts the mass spectrum of Pedunculagin.

FIG. 11 depicts the results of the screening assays for inhibition ofaggregation using Aβ₂₅₋₃₅ and ThT fluorescence with various fractionsisolated using different extraction solvents.

FIG. 12 depicts molecules identified in the extracts as bioactive aswell as their IC₅₀ values for inhibiting aggregation of Aβ₂₅₋₃₅.

FIG. 13 depicts the results using Punicalin in dose-responsefluorescence in vitro assays on Aβ₂₅₋₃₅.

FIG. 14 depicts the results using Pedunculagin in dose-responsefluorescence in vitro assays on Aβ₂₅₋₃₅.

FIG. 15 depicts the results using Punicalagin in dose-responsefluorescence in vitro assays on Aβ₂₅₋₃₅.

FIG. 16 depicts the results using Compound A (3I) in dose-responsefluorescence in vitro assays on Aβ₂₅₋₃₅.

FIG. 17 depicts the results using Tellimagrandin in dose-responsefluorescence in vitro assays on Aβ₂₅₋₃₅.

FIG. 18 depicts the results using Corilagin in dose-responsefluorescence in vitro assays on Aβ₂₅₋₃₅.

FIG. 19 depicts the results of the PC12 cells assays showing inhibitionof the neuronal toxicity induced by Aβ using a fraction from thepomegranate husk shown to contain Punicalagin (dosing at 500 μg/mL).

FIG. 20 depicts the HPLC (254 nm) profile of several pomegranateextracts of the invention.

FIG. 21 depicts β-amyloid induced cell death assay in PC12 cells.

FIG. 22 depicts the HPLC profiles of several subfractions of Extract1767. These profiles were obtained from a Varian analytic HPLC equippedwith a Diode Array detector (DAD) using a 250 mm×4.6 mm Ø-0.5 μm Varian“XRS C18” column. The solvent and gradient used are shown in FIG. 23.

FIG. 23 depicts the solvent and gradient used in to obtain the HLPCtraces; and a graph showing the bioactivity of 1767 subfractions on PC12cell survival against Aβ toxicity. All subfractions were assayed at 30μg/mL. Subfractions 1767-2 and 1767-3 exhibit the highest activities; asthey also display high similarities in their HPLC profile, they werepooled for further fractionation and analysis.

FIG. 24 depicts the result of the Morris Water Maze probe test at theend of a 3-month treatment period with a pomegranate extract 31008 in amouse AD model (administration beginning at four months).

FIG. 25 depicts the results of mouse brain section staining for diffuseamyloid plaques at the end of a 3-month treatment period with apomegranate extract 31008 in a mouse AD model (administration beginningat four months).

FIG. 26 depicts the result of the Morris Water Maze probe test at theend of a 3-month treatment period with punicalagin or pomegranateextract 31008 (at two different doses, low and high), extract 61109 andextract 71109, in a mouse AD model (administration beginning at twomonths).

FIG. 27 depicts results of a social recognition study with aged rats(wherein the extract is 31008).

FIG. 28 depicts results of a Morris Water Maze reversal test with agedrats (wherein the extract is 31008 and the dosing is 30 μg.mL).

FIG. 29 depicts Compound A.

FIG. 30 depicts a flowchart showing an ultra sound assisted sequentialpomegranate husk extraction by various solvent endowed with increasingpolarity; and HPLC profiles of the resulting extracts. These profileswere obtained from a Varian analytic HPLC equipped with a Diode ArrayDetector (DAD) using a 250×4.6 mm, Ø0.5 mm “XRS pursuit” diphenylcolumn. The solvent and gradient used are shown in FIG. 31.

FIG. 31 depicts the solvent and gradient used in to obtain the HLPCtraces; and a graph showing the bioactivity of husk subfractions on PC12cell survival against Aβ toxicity. All subfractions were assayed at 500μg/mL. Fraction 1776 exhibits the highest activity and very interestingHPLC profile, distinct of that of 1777 and 1778 whose major peaks looklike Punicalagin. This result may suggest that another compound,different from punicalagin may be able to protect PC12 cells fromAβ-induced toxicity. Therefore 1776 was selected for furtherfractionation. Tellimagrandin was identified in extract fraction 1776.

DETAILED DESCRIPTION

Certain aspects of the invention relate to compounds (as well asextracts and compositions containing the same) and methods for effectiveadministration of said compounds, extracts or compositions to a subjectin need thereof.

Amyloidosis

In certain embodiments, the compounds inhibit protein folding thatcauses Aβ peptide aggregation (i.e., amyloid plaque formation).

A number of incurable, ageing-related or degenerative diseases have beenlinked to a generic and fundamental pathogenic process of protein orpeptide misfolding and aggregation called “amyloidosis”. These includeAlzheimer's, Parkinson's and Huntington's diseases and type II diabetes.The amyloid deposits present in these diseases consist of particularpeptides that are characteristic for each of these diseases butregardless of their sequence the amyloid fibrils have a characteristicβ-sheet structure and share a common aggregation pathway. In eachdisease, a specific protein or peptide misfolds, adopts β-sheetstructure and oligomerizes to form soluble aggregation intermediates enroute to fibril formation ultimately forming insoluble amyloid fibres,plaques or inclusions. These insoluble forms of the aggregated proteinor peptide form by the intermolecular association of β-strands intoβ-sheets. Recent evidence suggests that the soluble amyloid oligomersmay be the principal cause of neurotoxicity.

The amyloidoses are defined as diseases in which normally solubleproteins accumulate in various tissues as insoluble deposits of fibrilsthat are rich in β-sheet structure and have characteristic dye-bindingproperties. Although the specific polypeptides that comprise thedeposits are different for each amyloidosis, the disorders have severalkey features in common. The most prominent of these is the ability ofproteins that are highly soluble in biological fluids to be graduallyconverted into insoluble filamentous polymers enriched in β-pleatedsheet conformation.

Furthermore, they tend to form by a similar molecular mechanism (by theintermolecular association of β-strands into extended β-sheets), so theytend to share a similar molecular structure and a common ability to bindcertain dyes, such as Congo Red and Thioflavin T.

These diseases and disorders, which are collectively referred to hereinas “amyloid-related diseases”, fall into two main categories: (a) thosewhich affect the brain and other parts of the central nervous system;and (b) those which affect other organs or tissues around the body.

Examples of amyloid-related diseases which fall under these twocategories are listed in the following two sections; however, many otherexamples of rare, hereditary amyloid-related diseases are known whichare not included here, and additional forms of amyloid-related diseaseare likely to be discovered in future.

Neurodegenerative Diseases Associated with Amyloidosis

Many different neurodegenerative diseases are associated with themisfolding and aggregation of a specific protein or peptide in aparticular part of the brain, or elsewhere in the central nervoussystem, depending on the specific disease. Examples of such diseasesfollow.

Various forms of Alzheimer's disease (AD) as well as Down's syndrome,hereditary cerebral hemorrhage with amyloidosis (HCHWA, Dutch type),cerebral amyloid angiopathy, and possibly also mild cognitive impairmentand other forms of dementia are associated with the aggregation of a40/42-residue peptide called β-amyloid, Aβ(1-40) or Aβ(1-42), whichforms insoluble amyloid fibres and plaques in the cerebral cortex,hippocampus or elsewhere in the brain, depending on the specificdisease. Alzheimer's disease is also associated with the formation ofneurofibrillary tangles by aggregation of a hyperphosphorylated proteincalled tau, which also occurs in frontotemporal dementia (Pick'sdisease).

Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiplesystem atrophy (MSA) are associated with the aggregation of a proteincalled α-synuclein, which results in the formation of insolubleinclusions called “Lewy bodies”. Huntington's disease (HD), spinal andbulbar muscular atrophy (SBMA, also known as Kennedy's disease),dentatorubral pallidoluysian atrophy (DRPLA), different forms ofspinocerebellar ataxia (SCA, types 1, 2, 3, 6 and 7), and possiblyseveral other inheritable neurodegenerative diseases are associated withthe aggregation of various proteins and peptides that contain abnormallyexpanded glutamine repeats (extended tracts of polyglutamine).Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE)in cows, scrapie in sheep, kuru, Gerstmann-Straussler-Scheinker disease(GSS), fatal familial insomnia, and possibly all other forms oftransmissible encephalopathy are associated with the self-propagatingmisfolding and aggregation of prion proteins.

Amyotrophic lateral sclerosis (ALS), and possibly also some other formsof motor neuron disease (MND) are associated with the aggregation of aprotein called superoxide dismutase.

Familial British dementia (FBD) and familial Danish dementia (FDD),respectively, are associated with aggregation of the ABri and ADanpeptide sequences derived from the BRI protein.

Hereditary cerebral hemorrhage with amyloidosis (HCHWA, Icelandic type)is associated with the aggregation of a protein called cystatin C.

Systemic Diseases Associated with Amyloidosis

In addition to the neurodegenerative diseases listed above, a widevariety of systemic ageing-related or degenerative diseases areassociated with the misfolding and aggregation of a particular proteinor peptide in various other tissues around the body (i.e., outside ofthe brain). Examples of such diseases follow.

Type II Diabetes (also known as adult-onset diabetes, or non-insulindependent diabetes mellitus) is associated with the aggregation of a37-residue peptide called the islet amyloid polypeptide (IAPP, or“amylin”), which forms insoluble deposits that are associated with theprogressive destruction of insulin-producing β cells in the islets ofLangerhans within the pancreas.

Dialysis-related amyloidosis (DRA) and prostatic amyloid are associatedwith the aggregation of a protein called β₂-microglobulin, either inbones, joints and tendons in DRA, which develops during prolongedperiods of hemodialysis, or within the prostate in the case of prostaticamyloid.

Primary systemic amyloidosis, systemic AL amyloidosis andmyeloma-associated amyloidosis are associated with the aggregation ofimmunoglobulin light chain (or in some cases immunoglobulin heavy chain)into insoluble amyloid deposits, which gradually accumulate in variousmajor organs such as the liver, kidneys, heart and gastrointestinal (GI)tract.

Reactive systemic AA amyloidosis, secondary systemic amyloidosis,familial Mediterranean fever and chronic inflammatory disease areassociated with the aggregation of serum amyloid A protein, which formsinsoluble amyloid deposits that accumulate in major organs such as theliver, kidneys and spleen; Senile systemic amyloidosis (SSA), familialamyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC)are associated with the misfolding and aggregation of different mutantsof transthyretin protein (TTR), which form insoluble inclusions invarious organs and tissues such as the heart (especially in FAC),peripheral nerves (especially in FAP) and gastrointestinal (GI) tract.Another form of familial amyloid polyneuropathy (FAP, type II) isassociated with the aggregation of apolipoprotein AI in the peripheralnerves; Familial visceral amyloidosis and hereditary non-neuropathicsystemic amyloidosis are associated with misfolding and aggregation ofvarious mutants of lysozyme, which form insoluble deposits in majororgans such as the liver, kidneys and spleen.

Finnish hereditary systemic amyloidosis is associated with aggregationof a protein called gelsolin in the eyes (particularly in the cornea).

Fibrinogen α-chain amyloidosis is associated with aggregation of thefibrinogen A α-chain, which forms insoluble amyloid deposits in variousorgans, such as the liver and kidneys.

Insulin-related amyloidosis occurs by the aggregation of insulin at thesite of injection in diabetics.

Medullary carcinoma of the thyroid is associated with the aggregation ofcalcitonin in surrounding tissues.

Isolated atrial amyloidosis is associated with the aggregation of atrialnatriuretic peptide (ANP) in the heart.

Various forms of cataract are associated with the aggregation ofγ-crystallin proteins in the lens of the eyes.

Pathogenic Mechanism of Amyloid-Related Diseases

While all of the amyloid-related diseases share the common pathogenicprocess of amyloidosis, the precise molecular mechanisms by which thisgeneric process of protein/peptide misfolding and aggregation is linkedto the progressive degeneration of affected tissues is unclear. In somecases, including many of the systemic amyloid-related diseases, it isthought that the sheer mass of insoluble protein or peptide simplyoverwhelms the affected tissues, ultimately leading to acute organfailure. In other cases, including most of the neurodegenerativediseases listed above, the symptoms of disease develop with theappearance of only very small aggregates. Therefore, it has beensuggested that the insoluble deposits are inherently toxic and mightcause the progressive destruction of cells, for example by causinginflammation and oxidative stress, or by directly interfering with cellmembranes or other cellular components or processes.

Recently, it has been established that the specific proteins andpeptides involved in at least some of these amyloid-related diseasesform various soluble oligomeric species during their aggregation, whichrange in size from dimers and trimers to much larger species comprisingtens or even hundreds or thousands of protein or peptide monomers.Moreover, the oligomers are inherently toxic to cells in vitro in theabsence of insoluble aggregates, and they appear to share a commonstructural feature as they can all be recognized by the same antibodydespite the fact that they may be formed by proteins or peptides withvery different amino acid sequences.

The molecular structure of these toxic soluble oligomers is not knownand the precise mechanism by which they kill cells is also unclear, butseveral theories have been proposed. According to one theory, called the“channel hypothesis,” the oligomers form heterogeneous pores or leakyion channels, which allow ions to flow freely through cell membranes,thereby destroying their integrity which ultimately causes cell death.Alternatively, or additionally, the oligomers may form protofibrils thatkill cells by a similar or different mechanism.

Regardless of the precise pathogenic mechanism, however, an overwhelmingamount of evidence has now been accumulated which suggests that thegeneral process of protein/peptide aggregation is the primary cause ofthese and possibly other amyloid-related diseases.

The present invention relates to chemical compounds, extracts andcompositions which are inhibitors of amyloid-related toxicity and, assuch, will be useful in the treatment of amyloid-related diseases anddisorders.

Early Detection of Amyloid-Related Diseases and Disorders

It is always desirable to detect diseases early in their progress. Earlydetection enables early treatment which has generally been proven toyield a higher success rate in treating various diseases. Recently, ithas been discovered that analyzing peoples' eyes, and in particular thelenses of the eyes, can yield indications of various types of diseases.For example, measurements taken of light scattering within the eye hasbeen shown to provide useful diagnostic information to detect andmonitor the progress of diseases such as Alzheimer's disease. US PatentApplication Publication No. 2008/0088795 and U.S. Pat. No. 7,107,092;both of which are hereby incorporated by reference in their entirety. Inaddition, changes in the brain measured with MRI and PET scans, ELISAassays, and diffraction-enhanced imaging (DEI), alone or combined withmemory tests and detection of risk proteins in body fluids, may alsolead to earlier and more accurate diagnosis of Alzheimer's.

Compounds of the Invention

One aspect of the invention relates to compounds s which whenadministered lead to the treatment or prevention of neurodegenerativedisorders and/or the improvement of brain health. As discussed in moredetail below, in certain embodiments the administration of the compoundresults in fewer depositions of amyloid fibrils in the brains of animalsfed compositions which comprise one or more compounds of the invention;and resulted in an increase or restoration of memory in the animals fedthe compositions comprising one or more compounds of the invention.

For example, compounds of the invention include Punicalin, Punicalagin,Pedunculagin, Tellimagrandin, Corilagin, Granatine A, Granatine B,Terminalin, Gallagyldilactone, and Compound A, as well aspharmaceutically acceptable salts, biologically active metabolites,solvates, hydrates, prodrugs, enantiomers or stereoisomers thereof.

The present invention also relates to a pure and isolated compound, or apharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof; whereinthe compound is represented by formula I:

wherein independently for each occurrence

X is

and

R¹, R² and R³ are each independently hydrogen, alkyl, aralkyl,alkylcarboxy, or a sugar; or

R¹ is hydrogen, alkyl, aralkyl, alkylcarboxy,

or a sugar, R² is hydrogen or

and R³ is

or

R¹ is hydrogen, alkyl, aralkyl, alkylcarboxy, or a sugar, and R² and R³taken

together are

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein X is

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein X is

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R¹ is hydrogen.

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R¹ is a sugar selected from the groupconsisting of allose (“All”), altrose (“Alt”), arabinose (“Ara”),erythrose, erythrulose, fructose (“Fru”), fucosamine (“FucN”), fucose(“Fuc”), galactosamine (“GalN”), galactose (“Gal”), galloyl-β-glucose,glucosamine (“GlcN”), glucosaminitol (“GlcN-ol”), glucose (“Glc”),glyceraldehyde, 2,3-dihydroxypropanal, glycerol (“Gro”),propane-1,2,3-triol, glycerone (“1,3-dihydroxyacetone”),1,3-dihydroxypropanone, gulose (“Gul”), idose (“Ido”), lyxose (“Lyx”),mannosamine (“ManN”), mannose (“Man”), psicose (“Psi”), quinovose(“Qui”), quinovosamine, rhamnitol (“Rha-ol”), rhamnosamine (“RhaN”),rhamnose (“Rha”), ribose (“Rib”), ribulose (“Rul”), rutinose, sialicacid (“Sia” or “Neu”), sorbose (“Sor”), tagatose (“Tag”), talose(“Tal”), tartaric acid, erythraric/threaric acid, threose, xylose(“Xyl”), or xylulose (“Xul”).

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R² is hydrogen.

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R² is a sugar selected from the groupconsisting of allose (“All”), altrose (“Alt”), arabinose (“Ara”),erythrose, erythrulose, fructose (“Fru”), fucosamine (“FucN”), fucose(“Fuc”), galactosamine (“GalN”), galactose (“Gal”), galloyl-β-glucose,glucosamine (“GlcN”), glucosaminitol (“GlcN-ol”), glucose (“Glc”),glyceraldehyde, 2,3-dihydroxypropanal, glycerol (“Gro”),propane-1,2,3-triol, glycerone (“1,3-dihydroxyacetone”),1,3-dihydroxypropanone, gulose (“Gul”), idose (“Ido”), lyxose (“Lyx”),mannosamine (“ManN”), mannose (“Man”), psicose (“Psi”), quinovose(“Qui”), quinovosamine, rhamnitol (“Rha-ol”), rhamnosamine (“RhaN”),rhamnose (“Rha”), ribose (“Rib”), ribulose (“Rul”), rutinose, sialicacid (“Sia” or “Neu”), sorbose (“Sor”), tagatose (“Tag”), talose(“Tal”), tartaric acid, erythraric/threaric acid, threose, xylose(“Xyl”), or xylulose (“Xul”).

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R³ is hydrogen.

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R³ is a sugar selected from the groupconsisting of allose (“All”), altrose (“Alt”), arabinose (“Ara”),erythrose, erythrulose, fructose (“Fru”), fucosamine (“FucN”), fucose(“Fuc”), galactosamine (“GalN”), galactose (“Gal”), galloyl-β-glucose,glucosamine (“GlcN”), glucosaminitol (“GlcN-ol”), glucose (“Glc”),glyceraldehyde, 2,3-dihydroxypropanal, glycerol (“Gro”),propane-1,2,3-triol, glycerone (“1,3-dihydroxyacetone”),1,3-dihydroxypropanone, gulose (“Gul”), idose (“Ido”), lyxose (“Lyx”),mannosamine (“ManN”), mannose (“Man”), psicose (“Psi”), quinovose(“Qui”), quinovosamine, rhamnitol (“Rha-ol”), rhamnosamine (“RhaN”),rhamnose (“Rha”), ribose (“Rib”), ribulose (“Rul”), rutinose, sialicacid (“Sia” or “Neu”), sorbose (“Sor”), tagatose (“Tag”), talose(“Tal”), tartaric acid, erythraric/threaric acid, threose, xylose(“Xyl”), or xylulose (“Xul”).

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R¹, R² and R³ are each independentlyhydrogen, alkyl, aralkyl, alkylcarboxy, or a sugar.

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R¹, R² and R³ are hydrogen.

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R¹ is hydrogen, alkyl, aralkyl,alkylcarboxy,

or a sugar, R² is hydrogen or

and R³ is

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R¹ is hydrogen, R² is

and R³ is

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R¹ is

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R¹ is

R² is hydrogen, and R³ is

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R¹ is hydrogen, alkyl, aralkyl,alkylcarboxy, or a sugar, and R² and R³ taken together are

In certain embodiments, the present invention relates to any one of theaforementioned compounds, wherein R¹ is hydrogen, and R² and R³ takentogether are

The invention also encompasses multimers of formula I, such as dimers inwhich two monomers of the formula are bonded as described in Reed, J.D., C. G. Krueger, et al. (2005). “MALDI-TOF mass spectrometry ofoligomeric food polyphenols.” Phytochemistry 66(18): 2248-63. The dimersare naturally occurring dimers of ellagitannins; their detailedstructures are not fully elucidated, but they contain the basicstructural backbone identified in this invention. Trimers, tetramers andlarger oligomers are also encompassed in the present invention. In otherwords, multimers with, for example, two, three, four, five, six, seven,eight, nine, ten, or more repeats are encompassed in the presentinvention.

Certain compounds of the invention which have acidic substituents mayexist as salts with pharmaceutically acceptable acids or bases. Many ofthe compounds of the invention may be provided as salts withpharmaceutically compatible counterions (i.e., pharmaceuticallyacceptable salts). A “pharmaceutically acceptable salt” means anynon-toxic salt that, upon administration to a recipient, is capable ofproviding, either directly or indirectly, a compound or a prodrug of acompound of this invention. A “pharmaceutically acceptable counterion”is an ionic portion of a salt that is not toxic when released from thesalt upon administration to a recipient. Pharmaceutically compatiblesalts may be formed with many acids, including but not limited tohydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms.

Acids commonly employed to form pharmaceutically acceptable saltsinclude inorganic acids such as hydrogen bisulfide, hydrochloric,hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well asorganic acids such as para-toluenesulfonic, salicylic, tartaric,bitartaric, ascorbic, maleic, besylic, fumaric, gluconic, glucuronic,formic, glutamic, methanesulfonic, ethanesulfonic, benzenesulfonic,lactic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric,benzoic and acetic acid, and related inorganic and organic acids. Suchpharmaceutically acceptable salts thus include sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,terephathalate, sulfonate, xylenesulfonate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate,.beta.-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate and the like salts. Exemplary pharmaceutically acceptable acidaddition salts include those formed with mineral acids such ashydrochloric acid and hydrobromic acid, and especially those formed withorganic acids such as maleic acid.

Suitable bases for forming pharmaceutically acceptable salts with acidicfunctional groups include, but are not limited to, hydroxides of alkalimetals such as sodium, potassium, and lithium; hydroxides of alkalineearth metal such as calcium and magnesium; hydroxides of other metals,such as aluminum and zinc; ammonia, and organic amines, such asunsubstituted or hydroxy-substituted mono-, di-, or trialkylamines;dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine;diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkylamines), such as mono-, bis-, or tris-(2-hydroxyethyl)amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N-dialkyl-N-(hydroxy alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl)amine, or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike.

Certain compounds of the invention and their salts may exist in morethan one crystal form and the present invention includes each individualcrystal form and mixtures thereof.

Certain compounds of the invention and their salts may also exist in theform of solvates, for example hydrates, and the present inventionincludes each solvate and mixtures thereof.

Certain compounds of the invention may contain one or more chiralcenters, and exist in different optically active forms. When compoundsof the invention contain one chiral center, the compounds exist in twoenantiomeric forms and the present invention includes both enantiomersand mixtures of enantiomers, such as racemic mixtures. The enantiomersmay be resolved by methods known to those skilled in the art, forexample by formation of diastereoisomeric salts which may be separated,for example, by crystallization; formation of diastereoisomericderivatives or complexes which may be separated, for example, bycrystallization, gas-liquid or liquid chromatography; selective reactionof one enantiomer with an enantiomer-specific reagent, for exampleenzymatic esterification; or gas-liquid or liquid chromatography in achiral environment, for example on a chiral support for example silicawith a bound chiral ligand or in the presence of a chiral solvent. Itwill be appreciated that where the desired enantiomer is converted intoanother chemical entity by one of the separation procedures describedabove, a further step may be used to liberate the desired enantiomericform. Alternatively, specific enantiomers may be synthesized byasymmetric synthesis using optically active reagents, substrates,catalysts or solvents, or by converting one enantiomer into the other byasymmetric transformation.

When a compound of the invention contains more than one chiral center,it may exist in diastereoisomeric forms. The diastereoisomeric compoundsmay be separated by methods known to those skilled in the art, forexample chromatography or crystallization and the individual enantiomersmay be separated as described above. The present invention includes eachdiastereoisomer of compounds of the invention and mixtures thereof.

Certain compounds of the invention may exist in different tautomericforms or as different geometric isomers, and the present inventionincludes each tautomer and/or geometric isomer of compounds of theinvention and mixtures thereof.

Certain compounds of the invention may exist in different stableconformational forms which may be separable. Torsional asymmetry due torestricted rotation about an asymmetric single bond, for example becauseof steric hindrance or ring strain, may permit separation of differentconformers. The present invention includes each conformational isomer ofcompounds of the invention and mixtures thereof.

Certain compounds of the invention may exist in zwitterionic form andthe present invention includes each zwitterionic form of compounds ofthe invention and mixtures thereof.

As used herein the term “pro-drug” refers to an agent which is convertedinto the parent drug in vivo by some physiological chemical process(e.g., a prodrug on being brought to the physiological pH is convertedto the desired drug form). Pro-drugs are often useful because, in somesituations, they may be easier to administer than the parent drug. Theymay, for instance, be bioavailable by oral administration whereas theparent drug is not. The prodrug may also have improved solubility inpharmacological compositions over the parent drug. An example, withoutlimitation, of a pro-drug would be a compound of the present inventionwherein it is administered as an ester (the “pro-drug”) to facilitatetransmittal across a cell membrane where water solubility is notbeneficial, but then it is metabolically hydrolyzed to the carboxylicacid once inside the cell where water solubility is beneficial.Pro-drugs have many useful properties. For example, a pro-drug may bemore water soluble than the ultimate drug, thereby facilitatingintravenous administration of the drug. A pro-drug may also have ahigher level of oral bioavailability than the ultimate drug. Afteradministration, the prodrug is enzymatically or chemically cleaved todeliver the ultimate drug in the blood or tissue.

Exemplary pro-drugs upon cleavage release the corresponding free acid,and such hydrolyzable ester-forming residues of the compounds of thisinvention include but are not limited to carboxylic acid substituents(e.g., —C(O)₂H or a moiety that contains a carboxylic acid) wherein thefree hydrogen is replaced by (C₁-C₄)alkyl, (C₂-C₁₂)alkanoyloxymethyl,(C₄-C₉)1-(alkanoyloxy)ethyl, 1-methyl-1-(alkanoyloxy)-ethyl having from5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbonatoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms,1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms,N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms,1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms,3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl,di-N,N—(C₁-C₂)alkylamino(C₂-C₃)alkyl (such as β-dimethylaminoethyl),carbamoyl-(C₁-C₂)alkyl, N,N-di(C₁-C₂)-alkylcarbamoyl-(C₁-C₂)alkyl andpiperidino-, pyrrolidino- or morpholino(C₂-C₃)alkyl.

Exemplary pro-drugs release an hydroxyl of a compound of the inventionwherein the free hydrogen of a hydroxyl is replaced by(C₁-C₆)alkanoyloxymethyl, 1-((C₁-C₆)alkanoyloxy)ethyl,1-methyl-1-((C₁-C₆)alkanoyloxy)ethyl, (C₁-C₆)alkoxycarbonyl-oxymethyl,N—(C₁-C₆)alkoxycarbonylamino-methyl, succinoyl, (C₁-C₆)alkanoyl,α-amino(C₁-C₄)alkanoyl, arylactyl and α-aminoacyl, orα-aminoacyl-α-aminoacyl wherein said α-aminoacyl moieties areindependently any of the naturally occurring L-amino acids found inproteins, —P(O)(OH)₂, —P(O)(O(C₁-C₆)alkyl)₂ or glycosyl (the radicalresulting from detachment of the hydroxyl of the hemiacetal of acarbohydrate).

A compound may be isolated and extracted (i.e., separated from thecompounds with which it naturally occurs), or it may be syntheticallyprepared (i.e., manufactured using a process synthesis) so that in anycases the level of contaminating compounds or impurities does notdetract from or adversely effect to the effectiveness of the compound ofthe invention.

Certain compounds of the invention may be extracted from natural sourcessuch as Punica granatum juice, leaves, bark, pericarp, or peel. Lansky,E. P. and R. A. Newman (2007). “Punica granatum (pomegranate) and itspotential for prevention and treatment of inflammation and cancer.” JEthnopharmacol 109(2): 177-206. Some of the compound may also beisolated from Walnuts, Euscaphis japonica, Geum japonicum Thunb. var.chinense, Blackberries (Rubus sp.), Juglans regia, Pimenta dioica,Quercus, Acer, Cornus officinalis, Emblica officinalis (amla),Terminalia chebula Retz, and Terminalia catappa L, as well as othersimilar species.

For example, Punicalin, Punicalagin, Pedunculagin and Tellimagrandinhave been previously isolated and characterized. Tanaka, K., G. Nonaka,et al. (1986). “Tannins and Related Compounds. XLI. 1) Isolation andCharactherization of Novel Ellagitannins, Punicacorteins A, B, C, andPunigluconin from the bark of Punica granatum L.” Chem. Pharm. Bull34(2): 656-663; Tanaka, K., G. Nonaka, et al. (1986). “Tannins andRelated Compounds. XLI. 1). Revision of the Structures of Punicalin andPunicalagin, and Isolation and Charactherization of 2-O-Galloylpunicalinfrom the Bark of Punica granatum L.” Chem. Pharm. Bull 34(2): 650-655;and Satomi, H., K. Umemura, et al. (1993). “Carbonic anhydraseinhibitors from the pericarps of Punica granatum L.” Biol Pharm Bull16(8): 787-90.

In certain embodiments, the compound of the invention is at least about1% pure up to about 99% pure. In certain embodiments, the compound ofthe invention is at least about 10% pure. In certain embodiments, thecompounds is at least about 20% pure. In certain embodiments, thecompounds is at least about 30% pure. In certain embodiments, thecompounds is at least about 40% pure. In certain embodiments, thecompounds is at least about 50% pure. In certain embodiments, thecompounds is at least about 60% pure. In certain embodiments, thecompounds is at least about 70% pure. In certain embodiments, thecompounds is at least about 80% pure. In certain embodiments, thecompounds is at least about 90% pure. In certain embodiments, thecompounds is at least about 95% pure. In certain embodiments, thecompounds is at least about 99% pure. In certain embodiments, suchextracts which contain one or more compounds of the invention at any ofthe aforementioned purities may be also suitable for use as or infunctional foods and dietary supplements. Such extracts are discussedmore fully below.

One aspect of the invention relates to a compound, or a pharmaceuticallyacceptable salt, biologically active metabolite, solvate, hydrate,prodrug, enantiomer or stereoisomer thereof, which generates the spectrain FIGS. 2 and 3.

One aspect of the invention relates to a compound, or a pharmaceuticallyacceptable salt, biologically active metabolite, solvate, hydrate,prodrug, enantiomer or stereoisomer thereof, which generates the spectrain FIGS. 4 and 5.

One aspect of the invention relates to a compound, or a pharmaceuticallyacceptable salt, biologically active metabolite, solvate, hydrate,prodrug, enantiomer or stereoisomer thereof, which generates the spectrain FIGS. 6 and 7.

One aspect of the invention relates to a compound, or a pharmaceuticallyacceptable salt, biologically active metabolite, solvate, hydrate,prodrug, enantiomer or stereoisomer thereof, which generates the spectrain FIGS. 8 and 9.

One aspect of the invention relates to a compound, or a pharmaceuticallyacceptable salt, biologically active metabolite, solvate, hydrate,prodrug, enantiomer or stereoisomer thereof, which generates thespectrum in FIG. 10.

In certain embodiments, the present invention relates to any of theaforementioned compounds, wherein said compound, or a pharmaceuticallyacceptable salt, biologically active metabolite, solvate, hydrate,prodrug, enantiomer or stereoisomer thereof, was prepared by extractionfrom a plant material, as described herein. For example, one aspect ofthe invention relates to a compound prepared by a process comprising thesteps of using an adsorbent to bind said compound; and eluting the boundcompound from the adsorbent.

Isolation/Preparation

As noted above, extracts enriched in one or more compounds of theinvention may be recovered from plant material, which plant material mayinclude fruit, husks, juice, leaves, woody stems, and the like. Plantssuitable for extraction include Punica granatum. Methods of purificationare described in US Patent Application Publication No. US 2008/0318877to Seeram et al., which is hereby incorporated by reference in itsentirety.

A suspension of material from the plant material may be prepared by avariety of methods as known in the art, e.g., blending and aqueousextraction. The plant material may be subjected to enzymatic treatmentincluding but not limited to extractase, pectinase and the like. Theaqueous solution comprising compounds of the invention is applied to apolymeric adsorbent column, which is then washed with an aqueous bufferto remove unbound material. The compounds of interest bind to the resin,and may be eluted with a polar solvent, e.g., water, ethanol, methanol,and acetone.

The resin has a surface to which the compounds are adsorbed. A class ofadsorptive resins are polymeric crosslinked resins composed of styreneand divinylbenzene such as, for example, the AMBERLITE series of resins,e.g., AMBERLITE XAD-16, which are available commercially from Rohm &Haas Co., Philadelphia, Pa. Other polymeric crosslinked styrene anddivinylbenzene adsorptive resins suitable for use according to theinvention are XFS-4257, XFS-4022, XUS-40323 and XUS-40322, allmanufactured by The Dow Chemical Company, Midland, Mich.

One may use commercially available, FDA-approved,styrene-divinyl-benzene (SDVB) cross-linked copolymer resin, (e.g.,AMBERLITE XAD-16, as described in U.S. Pat. No. 4,297,220, hereinincorporated by reference). This resin is a non-ionic hydrophobic,cross-linked polystyrene divinyl benzene adsorbent resin. AMBERLITEXAD-16 has a macroreticular structure, with both a continuous polymerphase and a continuous pore phase. In certain embodiments, the resinused in the present invention has a particle size ranging from 100-200microns.

Other adsorbents, such as those in the AMBERLITE XAD adsorbent serieswhich contain hydrophobic macroreticular resin beads, with particlesizes in the range of 100-200 microns, may also be effective in themethods of the present invention. Moreover, different variations of theAMBERLITES, such as the AMERCHROM CG series of adsorbents, used withparticle sizes in the range of 100-200 microns, may also be suitable foruse in the present invention.

The resins are washed, e.g., with water or an aqueous buffer to removeunbound material from the extract. A solvent can be used to remove theadsorbed compounds, such as ethyl acetate or butanol.

The eluted compounds are substantially purified relative to the startingmaterial, and may be further purified, e.g., by chromatography, etc., ormay be directly used in formulations of interest. The final compositionmay be enriched, filtered, dialyzed, etc., using methods known in theart.

In certain embodiments, the present invention provides a compound of theinvention, or a pharmaceutically acceptable salt, biologically activemetabolite, solvate, hydrate, prodrug, enantiomer or stereoisomerthereof, substantially free of other compounds found in the plantmaterial from which it is extracted. As used herein, the term“substantially free” means that the compound is made up of asignificantly greater proportion of a compound of the invention, or apharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, ascompared with the compound as found in the plant material from which itis extracted or extracts thereof.

In some embodiments, the present invention provides a compound of theinvention, or a pharmaceutically acceptable salt, biologically activemetabolite, solvate, hydrate, prodrug, enantiomer or stereoisomerthereof, in an amount of about 1 weight percent to about 99 weightpercent.

In certain embodiments, the compound of the invention is provided ingreater than about 2% chemical purity. In certain embodiments, thecompound of the invention is provided in greater than about 10% chemicalpurity. In certain embodiments, the compound of the invention isprovided in greater than about 20% chemical purity. In certainembodiments, the compound of the invention is provided in greater thanabout 30% chemical purity. In certain embodiments, the compound of theinvention is provided in greater than about 40% chemical purity. Incertain embodiments, the compound of the invention is provided ingreater than about 50% chemical purity. In certain embodiments, thecompound of the invention is provided in greater than about 75% chemicalpurity. In certain embodiments, the compound of the invention isprovided in greater than about 80% chemical purity. In certainembodiments, the compound of the invention is provided in greater thanabout 85% chemical purity. In certain embodiments, the compound of theinvention is provided in greater than about 90% chemical purity.

In certain embodiments, the compound of the invention is provided atbetween about 2% chemical purity and 10% chemical purity. In certainembodiments, the compound of the invention is provided at between about10% chemical purity and 30% chemical purity. In certain embodiments, thecompound of the invention is provided at between about 20% chemicalpurity and about 40% chemical purity. In certain embodiments, thecompound of the invention is provided at between about 30% chemicalpurity and about 50% chemical purity. In certain embodiments, thecompound of the invention is provided at between about 40% chemicalpurity and about 60% chemical purity. In certain embodiments, thecompound of the invention is provided at between about 50% chemicalpurity and about 70% chemical purity. In certain embodiments, thecompound of the invention is provided at between about 75% chemicalpurity and 95% chemical purity.

In other embodiments, the compound of the invention, or apharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, containsno more than about 50 area percent HPLC of other components of the plantmaterial from which it is extracted relative to the total area of theHPLC chromatogram. In other embodiments, the compound of the invention,or a pharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, containsno more than about 25 area percent HPLC of other components of the plantmaterial from which it is extracted relative to the total area of theHPLC chromatogram. In other embodiments, the compound of the invention,or a pharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, containsno more than about 20 area percent HPLC of other components of the plantmaterial from which it is extracted relative to the total area of theHPLC chromatogram. In other embodiments, the compound of the invention,or a pharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, containsno more than about 15 area percent HPLC of other components of the plantmaterial from which it is extracted relative to the total area of theHPLC chromatogram. In other embodiments, the compound of the invention,or a pharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, containsno more than about 10 area percent HPLC of other components of the plantmaterial from which it is extracted relative to the total area of theHPLC chromatogram. In other embodiments, the compound of the invention,or a pharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, containsno more than about 9 area percent HPLC of other components of the plantmaterial from which it is extracted relative to the total area of theHPLC chromatogram. In other embodiments, the compound of the invention,or a pharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, containsno more than about 8 area percent HPLC of other components of the plantmaterial from which it is extracted relative to the total area of theHPLC chromatogram. In other embodiments, the compound of the invention,or a pharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, containsno more than about 7 area percent HPLC of other components of the plantmaterial from which it is extracted relative to the total area of theHPLC chromatogram. In other embodiments, the compound of the invention,or a pharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, containsno more than about 6 area percent HPLC of other components of the plantmaterial from which it is extracted relative to the total area of theHPLC chromatogram. In other embodiments, the compound of the invention,or a pharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, containsno more than about 5 area percent HPLC of other components of the plantmaterial from which it is extracted relative to the total area of theHPLC chromatogram. In certain embodiments, said plant source is Punicagranatum.

In addition, compounds of the present invention may be prepared bysemi-synthetic processes starting from other compounds found inextracts. This may be accomplished either by chemical or biologicaltransformation of an isolated compound or an extract fraction or mixtureof compounds. Chemical transformation may be accomplished by, but notlimited to, manipulation of temperature, pH, and/or treatment withvarious solvents. Biological transformation may be accomplished by, butnot limited to, treatment of an isolated compound or an extract fractionor mixture of compounds with plant tissue, plant tissue extracts, othermicrobiological organisms or an isolated enzyme from any organism.

Extracts

One aspect of the present invention relates to an extract comprising anaforementioned compound or compounds, or pharmaceutically acceptablesalts, biologically active metabolites, solvates, hydrates, prodrugs,enantiomers or stereoisomers thereof.

Another aspect of the present invention relates to an extract comprisingbetween about 1% to about 10% of an aforementioned compound orcompounds, or pharmaceutically acceptable salts, biologically activemetabolites, solvates, hydrates, prodrugs, enantiomers or stereoisomersthereof.

Another aspect of the present invention relates to an extract comprisingbetween about 10% to about 20% of an aforementioned compound orcompounds, or pharmaceutically acceptable salts, biologically activemetabolites, solvates, hydrates, prodrugs, enantiomers or stereoisomersthereof.

Another aspect of the present invention relates to an extract comprisingbetween about 20% to about 30% of an aforementioned compound orcompounds, or pharmaceutically acceptable salts, biologically activemetabolites, solvates, hydrates, prodrugs, enantiomers or stereoisomersthereof.

Another aspect of the present invention relates to an extract orcomposition comprising between about 30% to about 40% of anaforementioned compound or compounds, or pharmaceutically acceptablesalts, biologically active metabolites, solvates, hydrates, prodrugs,enantiomers or stereoisomers thereof.

Another aspect of the present invention relates to an extract orcomposition comprising between about 40% to about 50% of anaforementioned compound or compounds, or pharmaceutically acceptablesalts, biologically active metabolites, solvates, hydrates, prodrugs,enantiomers or stereoisomers thereof.

Another aspect of the present invention relates to an extract orcomposition comprising between about 50% to about 60% of anaforementioned compound or compounds, or pharmaceutically acceptablesalts, biologically active metabolites, solvates, hydrates, prodrugs,enantiomers or stereoisomers thereof.

Another aspect of the present invention relates to an extract orcomposition comprising between about 60% to about 70% of anaforementioned compound or compounds, or pharmaceutically acceptablesalts, biologically active metabolites, solvates, hydrates, prodrugs,enantiomers or stereoisomers thereof.

Another aspect of the present invention relates to an extract orcomposition comprising between about 70% to about 80% of anaforementioned compound or compounds, or pharmaceutically acceptablesalts, biologically active metabolites, solvates, hydrates, prodrugs,enantiomers or stereoisomers thereof.

Another aspect of the present invention relates to an extract orcomposition comprising between about 80% to about 90% of anaforementioned compound or compounds, or pharmaceutically acceptablesalts, biologically active metabolites, solvates, hydrates, prodrugs,enantiomers or stereoisomers thereof.

Another aspect of the present invention relates to an extract orcomposition comprising between about 90% to about 95% of anaforementioned compound or compounds, or pharmaceutically acceptablesalts, biologically active metabolites, solvates, hydrates, prodrugs,enantiomers or stereoisomers thereof.

Another aspect of the present invention relates to an extract orcomposition comprising between about 95% to about 99% of anaforementioned compound or compounds, or pharmaceutically acceptablesalts, biologically active metabolites, solvates, hydrates, prodrugs,enantiomers or stereoisomers thereof.

In certain embodiments, said extract is a Punica granatum extract.

Compositions

The compounds and extracts of the invention may be used to formulatepharmaceuticals, nutraceuticals, botanical drugs, herbal medicines, foodadditive, functional foods, medical foods, nutrition products,cosmetics, beverages, and the like.

The compounds and/or extracts of the invention may be provided as acomposition with a pharmaceutically acceptable carrier. Such dosageforms encompass physiologically acceptable carriers that are inherentlynon-toxic and non-therapeutic. Examples of such carriers includevegetable proteins, soy proteins, ion exchangers, soft gels, oils,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts, or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, and PEG. Carriers fortopical or gel-based forms include polysaccharides such as sodiumcarboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, PEG, andwood wax alcohols. For all administrations, conventional depot forms aresuitably used. Such forms include, for example, microcapsules,nano-capsules, liposomes, plasters, inhalation forms, nose sprays,sublingual tablets, and sustained-release preparations.

In certain embodiments, the compounds and/or extracts may be formulatedin such vehicles at a concentration of about 0.01 μg/mL to about 200mg/mL. In certain embodiments, the compounds and/or extracts may beformulated in such vehicles at a concentration of about 1 μg/mL to about250 μg/mL. In certain embodiments, the compounds and/or extracts may beformulated in such vehicles at a concentration of about 250 μg/mL toabout 500 μg/mL. In certain embodiments, the compounds and/or extractsmay be formulated in such vehicles at a concentration of about 500 μg/mLto about 750 μg/mL. In certain embodiments, the compounds and/orextracts may be formulated in such vehicles at a concentration of about750 μg/mL to about 1 mg/mL. In certain embodiments, the compounds and/orextracts may be formulated in such vehicles at a concentration of about1 mg/mL to about 25 mg/mL. T In certain embodiments, the compoundsand/or extracts may be formulated in such vehicles at a concentration ofabout 25 mg/mL to about 50 mg/mL. In certain embodiments, the compoundsand/or extracts may be formulated in such vehicles at a concentration ofabout 50 mg/mL to about 100 mg/mL. In certain embodiments, the compoundsand/or extracts may be formulated in such vehicles at a concentration ofabout 100 mg/mL to about 125 mg/mL. In certain embodiments, thecompounds and/or extracts may be formulated in such vehicles at aconcentration of about 125 mg/mL to about 150 mg/mL. In certainembodiments, the compounds and/or extracts may be formulated in suchvehicles at a concentration of about 150 mg/mL to about 175 mg/mL. Incertain embodiments, the compounds and/or extracts may be formulated insuch vehicles at a concentration of about 175 mg/mL to about 200 mg/mL.

In certain embodiments, compounds, compositions and/or extracts of theinvention may be combined with herbal medicines. Herbal medicines ofinterest include, but not restricted to, active fractions from certainherbal preparations, such as nettles (Urtica dioica)-turmeric (Curcumalonga), tea; marine or terrestrial animal products, e.g., bioactivelipids from Perna canaliculus, or Dromaius nova hollandiae.

In certain embodiments, compounds, compositions and/or extracts of theinvention may be formulated as botanical drugs. As used herein, a“botanical drug” is a product consists of vegetable materials, which mayinclude plant materials, algae, macroscopic fungi, or combinationsthereof, which is intended for use in the diagnosis, cure, mitigation,treatment or prevention of disease in humans. In certain embodiments,the botanical drug product may be available as (but not limited to) asolution (e.g., tea), powder, tablet, capsule, elixir, topical, orinjection.

In certain embodiments, compounds, compositions and/or extracts of theinvention may be formulated as nutraceuticals. Nutraceuticalformulations of interest include foods for veterinary or human use,including health food bars, drinks and drink supplements, and the like.These foods are enhanced by the inclusion of a biologically activecompound, composition and/or extract of the invention. For example, inthe treatment of neurodegenerative diseases, such as Alzheimer's, thenormal diet of a patient may be supplemented by a nutraceuticalformulation taken on a regular basis. Such nutraceuticals may or may notcontain calories.

The term nutraceutical composition as used herein include food product,foodstuff, dietary supplement, nutritional supplement or a supplementcomposition for a food product or a foodstuff. Thus, in anotherembodiment the present invention relates to a nutraceutical wherein thenutraceutical is a food product, foodstuff, dietary supplement,nutritional supplement or a supplement composition for a food product ora foodstuff.

As used herein, the term food product refers to any food or feedsuitable for consumption by humans or animals. The food product may be aprepared and packaged food (e.g., mayonnaise, salad dressing, bread, orcheese) or an animal feed (e.g., extruded and pelleted animal feed,coarse mixed feed or pet food composition). As used herein, the termfoodstuff refers to any substance fit for human or animal consumption.Food products or foodstuffs are for example beverages such asnon-alcoholic and alcoholic drinks as well as liquid preparation to beadded to drinking water and liquid food, non-alcoholic drinks are forinstance soft drinks, sport drinks, fruit juices, such as for exampleorange juice, apple juice and grapefruit juice; lemonades, teas,near-water drinks, milk, milk replacements, and other dairy drinks suchas for example yoghurt drinks, and diet drinks. In another embodimentfood products or foodstuffs refer to solid or semi-solid foodscomprising the composition according to the invention. These forms caninclude, but are not limited to baked goods such as bars, cakes,cookies, puddings, dairy products, confections, snack foods, or frozenconfections or novelties (e.g., ice cream, milk shakes), prepared frozenmeals, candy, snack products (e.g., chips), liquid food such as soups,spreads, sauces, salad dressings, prepared meat products, cheese,yoghurt and any other fat or oil containing foods, and food ingredients(e.g., wheat flour). The term food products or foodstuffs also includesfunctional foods and prepared food products, the latter referring to anypre-packaged food approved for human consumption.

In certain embodiments, the nutraceutical formulation may furthercomprise curcumin or tea catechins, such as EGCG, L-theanines, andresveratrol. In certain embodiments, the nutraceutical formulation maycomprise extracts of acai berry, blueberry, cranberry, blackberry,raspberry, elderberry, St-Johns Wort, ginkgo biloba, kava, cocoa, winegrapes, grape seeds extracts, soy extracts, soy phytoestrogens, orcombinations thereof.

In certain embodiments, compounds, compositions and/or extracts of theinvention may be formulated as dietary supplements. Dietary supplementsof the present invention may be delivered in any suitable format. Incertain embodiments, dietary supplements are formulated for oraldelivery. The ingredients of the dietary supplement of this inventionare contained in acceptable excipients and/or carriers for oralconsumption. The actual form of the carrier, and thus, the dietarysupplement itself, is not critical. The carrier may be a liquid, gel,gelcap, capsule, powder, solid tablet (coated or non-coated), tea, orthe like. The dietary supplement is preferably in the form of a tabletor capsule and most preferably in the form of a hard (shell) gelatincapsule. Suitable excipient and/or carriers include maltodextrin,calcium carbonate, dicalcium phosphate, tricalcium phosphate,microcrystalline cellulose, dextrose, rice flour, magnesium stearate,stearic acid, croscarmellose sodium, sodium starch glycolate,crospovidone, sucrose, vegetable gums, lactose, methylcellulose,povidone, carboxymethylcellulose, corn starch, and the like (includingmixtures thereof). Exemplary carriers include calcium carbonate,magnesium stearate, maltodextrin, and mixtures thereof. The variousingredients and the excipient and/or carrier are mixed and formed intothe desired form using conventional techniques. The tablet or capsule ofthe present invention may be coated with an enteric coating thatdissolves at a pH of about 6.0 to 7.0. A suitable enteric coating thatdissolves in the small intestine but not in the stomach is celluloseacetate phthalate. Further details on techniques for formulation for andadministration may be found in the latest edition of Remington'sPharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

In other embodiments, the dietary supplement is provided as a powder orliquid suitable for adding by the consumer to a food or beverage. Forexample, in some embodiments, the dietary supplement can be administeredto an individual in the form of a powder, for instance to be used bymixing into a beverage, or by stirring into a semi-solid food such as apudding, topping, sauce, puree, cooked cereal, or salad dressing, forinstance, or by otherwise adding to a food; e.g., enclosed in caps offood or beverage containers for release immediately before consumption.The dietary supplement may comprise one or more inert ingredients,especially if it is desirable to limit the number of calories added tothe diet by the dietary supplement. For example, the dietary supplementof the present invention may also contain optional ingredientsincluding, for example, herbs, vitamins, minerals, enhancers, colorants,sweeteners, flavorants, inert ingredients, and the like.

In some embodiments, the dietary supplements further comprise vitaminsand minerals including, but not limited to, calcium phosphate oracetate, tribasic; potassium phosphate, dibasic; magnesium sulfate oroxide; salt (sodium chloride); potassium chloride or acetate; ascorbicacid; ferric orthophosphate; niacin, niacinamide; zinc sulfate or oxide;calcium pantothenate; copper gluconate; riboflavin; beta-carotene;pyridoxine hydrochloride; thiamin mononitrate; folic acid; biotin;chromium chloride or picolonate; potassium iodide; sodium selenate;sodium molybdate; phylloquinone; vitamin D3; cyanocobalamin; sodiumselenite; copper sulfate; vitamin A; vitamin C; inositol; potassiumiodide; L-tryptophan; nicotinic acid; nicotinamide; nicotinamideriboside; omega-3 fatty acid (such as DHA, EPA and ALA); anthocyanines;isoflavones; choline; UMP; soy phospholipids; phosphatidyl serine;S-adenosyl-methionine (SAM); acethyl-L-carnitine (ALCAR); magnesiumsalts; magnesium acetate; magnesium chloride; magnesium citrate;magnesium lactate; magnesium gluconante; and magnesium pidolate.

In other embodiments, the present invention provides nutritionalsupplements (e.g., energy bars or meal replacement bars or beverages)comprising a compound, composition and/or extract according to theinvention. The nutritional supplement may serve as meal or snackreplacement and generally provide nutrient calories. However, as notedabove, supplements which do not contain calories may also be used. Incertain embodiments, the nutritional supplements provide carbohydrates,proteins, and fats in balanced amounts. The nutritional supplement canfurther comprise carbohydrate, simple, medium chain length, orpolysaccharides, or a combination thereof. A simple sugar can be chosenfor desirable organoleptic properties. Uncooked cornstarch is oneexample of a complex carbohydrate. If it is desired that it shouldmaintain its high molecular weight structure, it should be included onlyin food formulations or portions thereof which are not cooked or heatprocessed since the heat will break down the complex carbohydrate intosimple carbohydrates, wherein simple carbohydrates are mono- ordisaccharides. The nutritional supplement contains, in one embodiment,combinations of sources of carbohydrate of three levels of chain length(simple, medium and complex; e.g., sucrose, maltodextrins, and uncookedcornstarch).

Sources of protein to be incorporated into the nutritional supplement ofthe invention can be any suitable protein utilized in nutritionalformulations and can include whey protein, whey protein concentrate,whey powder, egg, soy flour, soy milk soy protein, soy protein isolate,caseinate (e.g., sodium caseinate, sodium calcium caseinate, calciumcaseinate, potassium caseinate), animal and vegetable protein andhydrolysates or mixtures thereof. Soy protein have an almost perfectPDCAA, Protein Digestibility Corrected Amino Acid Score (PDCAAS) and bythis criterion soy protein is the nutritional equivalent of meat andeggs for human growth and health. These proteins have high biologicalvalue; that is, they have a high proportion of the essential aminoacids. See Modern Nutrition in Health and Disease, eighth edition, Lea &Febiger, publishers, 1986, especially Volume 1, pages 30-32. Thenutritional supplement can also contain other ingredients, such as oneor a combination of other vitamins, minerals, antioxidants, fiber andother dietary supplements (e.g., protein, amino acids, choline,lecithin, omega-3 fatty acids, and others discussed herein). Selectionof one or several of these ingredients is a matter of formulation,design, consumer preference and end-user. The amounts of theseingredients added to the dietary supplements of this invention arereadily known to the skilled artisan. Further vitamins and minerals thatcan be added include, but are not limited to, calcium phosphate oracetate, tribasic; potassium phosphate, dibasic; magnesium sulfate oroxide; salt (sodium chloride); potassium chloride or acetate; ascorbicacid; ferric orthophosphate; anthocyanins; nicotinamide riboside;magnesium salts; nicotinamide; zinc sulfate or oxide; calciumpantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxinehydrochloride; thiamin mononitrate; folic acid; biotin; chromiumchloride or picolonate; potassium iodide; sodium selenate; sodiummolybdate; phylloquinone; vitamin D3; cyanocobalamin; sodium selenite;copper sulfate; vitamin A; vitamin C; inositol; potassium iodide.

The nutritional supplement can be provided in a variety of forms, and bya variety of production methods. In one embodiment, to manufacture afood bar, the liquid ingredients are cooked; the dry ingredients areadded with the liquid ingredients in a mixer and mixed until the doughphase is reached; the dough is put into an extruder, and extruded; theextruded dough is cut into appropriate lengths; and the product iscooled. The bars may contain other nutrients and fillers to enhancetaste, in addition to the ingredients specifically listed herein.

It is understood by those of skill in the art that other ingredients canbe added to those described herein, for example, fillers, emulsifiers,preservatives, for the processing or manufacture of a nutritionalsupplement.

Additionally, flavors, coloring agents, spices, nuts and the like may beincorporated into the formulations described herein. Flavorings can bein the form of flavored extracts, volatile oils, chocolate flavorings,peanut butter flavoring, cookie crumbs, crisp rice, vanilla or anycommercially available flavoring. Examples of useful flavoring include,but are not limited to, pure anise extract, imitation banana extract,imitation cherry extract, chocolate extract, pure lemon extract, pureorange extract, pure peppermint extract, imitation pineapple extract,imitation rum extract, imitation strawberry extract, or pure vanillaextract; or volatile oils, such as balm oil, bay oil, bergamot oil,cedarwood oil, walnut oil, cherry oil, cinnamon oil, clove oil, orpeppermint oil.

Emulsifiers may be added for stability of the formulations. Examples ofsuitable emulsifiers include, but are not limited to, lecithin (e.g.,from egg or soy), and/or mono- and di-glycerides. Other emulsifiers arereadily apparent to the skilled artisan and selection of suitableemulsifier(s) will depend, in part, upon the formulation and finalproduct. Preservatives may also be added to the formulations to extendproduct shelf life. For example, preservatives such as potassiumsorbate, sodium sorbate, potassium benzoate, sodium benzoate or calciumdisodium EDTA are used.

In addition to the carbohydrates described above, the formulations cancontain natural or artificial (preferably low calorie) sweeteners, e.g.,saccharides, cyclamates, aspartamine, aspartame, acesulfame K, and/orsorbitol. Such artificial sweeteners can be desirable if the nutritionalsupplement is intended to be consumed by an overweight or obeseindividual, or an individual with type II diabetes who is prone tohyperglycemia.

Moreover, a multi-vitamin and mineral supplement may be added to theformulations of the present invention to obtain an adequate amount of anessential nutrient, which is missing in some diets. The multi-vitaminand mineral supplement may also be useful for disease prevention andprotection against nutritional losses and deficiencies due to lifestylepatterns.

The dosage and ratios of the compound(s) of the invention administeredvia a such formulations will, of course, vary depending upon knownfactors, such as the physiological characteristics of the particularcomposition and its mode and route of administration; the age, healthand weight of the recipient; the nature and extent of the symptoms; thekind of concurrent treatment; the frequency of treatment; and the effectdesired which can be determined by the expert in the field with normaltrials, or with the usual considerations regarding the formulation of anutraceutical composition.

In certain embodiments, the formulation comprises per serving an amountof 1 mg to 10,000 mg of the active ingredient(s), e.g., a compound orcompounds of the invention. In certain embodiments, the formulationcomprises per serving an amount of 1 mg to 2,500 mg of the activeingredient(s). In certain embodiments, the formulation comprises perserving an amount of 2500 mg to 5000 mg of the active ingredient(s). Incertain embodiments, the formulation comprises per serving an amount of5000 mg to 7500 mg of the active ingredient(s). In certain embodiments,the formulation comprises per serving an amount of 7500 mg to 10000 mgof the active ingredient(s).

For cosmetic formulations, the compounds, compositions and/or extractsof the invention may optionally comprise skin benefit materials. Theseinclude estradiol; progesterone; pregnanalone; coenzyme Q10;methylsolanomethane (MSM); copper peptide (copper extract); planktonextract (phytosome); glycolic acid; kojic acid; ascorbyl palmitate;all-trans-retinol; azaleic acid; salicylic acid; broparoestrol; estrone;adrostenedione; androstanediols; etc. The steroids will generally bepresent at a concentration of less than about 2% of the total by weightof the composition, while the other skin benefit materials may bepresent at higher levels, for example as much as about 10 to 15%.

The compounds, compositions and/or extracts of the invention maycomprise a cosmetically acceptable vehicle to act as a dilutant,dispersant or carrier, so as to facilitate its distribution when thecomposition is applied to the skin. Vehicles other than or in additionto water can include liquid or solid emollients, solvents, humectants,thickeners and powders.

The cosmetically acceptable vehicle will usually constitute from 0.1%,or 5% to 99.9%, preferably from 25% to 80% by weight of the composition,and can, in the absence of other cosmetic adjuncts, constitute thebalance of the composition.

In accordance with the present invention, a compound, composition and/orextract of the present invention may be prepared as pharmaceuticalcompositions, such as those which may be particularly useful for thetreatment of neurodegenerative diseases. Such compositions comprise acompound of the present invention or a pharmaceutically acceptable salt,biologically active metabolite, solvate, hydrate, prodrug, enantiomer orstereoisomer thereof, and a pharmaceutically acceptable carrier and/orexcipient.

The compositions of the present invention may be administered by variousmeans, depending on their intended use, as is well known in the art. Forexample, if compositions of the present invention are to be administeredorally, they may be formulated as tablets, capsules, granules, powdersor syrups. Alternatively, formulations of the present invention may beadministered parenterally as injections (intravenous, intramuscular orsubcutaneous), drop infusion preparations or suppositories. Forapplication by the ophthalmic mucous membrane route, compositions of thepresent invention may be formulated as eye drops or eye ointments. Theseformulations may be prepared by conventional means, and, if desired, thecompositions may be mixed with any conventional additive, such as anexcipient, a binder, a disintegrating agent, a lubricant, a corrigent, asolubilizing agent, a suspension aid, an emulsifying agent or a coatingagent.

In formulations of the subject invention, wetting agents, emulsifiersand lubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants may bepresent in the formulated agents.

Subject formulations may be suitable for oral, nasal, topical (includingbuccal and sublingual), rectal, vaginal, aerosol and/or parenteraladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of composition that may be combined with a carriermaterial to produce a single dose vary depending upon the subject beingtreated, and the particular mode of administration.

Methods of preparing these formulations include the step of bringinginto association compositions of the present invention with the carrierand, optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation agents with liquid carriers, or finely divided solidcarriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), each containing a predetermined amount of a subjectcomposition thereof as an active ingredient. Compositions of the presentinvention may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the subject composition ismixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, acetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingexcipients such as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the subject compositionmoistened with an inert liquid diluent. Tablets, and other solid dosageforms, such as dragees, capsules, pills and granules, may optionally bescored or prepared with coatings and shells, such as enteric coatingsand other coatings well known in the pharmaceutical-formulating art.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the subject composition, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Suspensions, in addition to the subject composition, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing a subject composition withone or more suitable non-irritating excipients or carriers comprising,for example, cocoa butter, polyethylene glycol, a suppository wax or asalicylate, and which is solid at room temperature, but liquid at bodytemperature and, therefore, will melt in the body cavity and release theactive agent. Formulations which are suitable for vaginal administrationalso include pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate.

Dosage forms for transdermal administration of a subject compositionincludes powders, sprays, ointments, pastes, creams, lotions, gels,solutions, patches and inhalants. The active component may be mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to asubject composition, excipients, such as animal and vegetable fats,oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays may contain, in addition to a subject composition,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays may additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Compositions of the present invention may alternatively be administeredby aerosol. This is accomplished by preparing an aqueous aerosol,liposomal preparation or solid particles containing the compound. Anon-aqueous (e.g., fluorocarbon propellant) suspension could be used.Sonic nebulizers may be used because they minimize exposing the agent toshear, which may result in degradation of the compounds contained in thesubject compositions.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of a subject composition together withconventional pharmaceutically acceptable carriers and stabilizers. Thecarriers and stabilizers vary with the requirements of the particularsubject composition, but typically include non-ionic surfactants(Tweens, Pluronics®, or polyethylene glycol), innocuous proteins likeserum albumin, sorbitan esters, oleic acid, lecithin, amino acids suchas glycine, buffers, salts, sugars or sugar alcohols. Aerosols generallyare prepared from isotonic solutions.

Compositions of this invention suitable for parenteral administrationcomprise a subject composition in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the compositions of the invention include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

In certain embodiments, the compositions are formulated as a tablet,pill capsule or other appropriate ingestible formulation, to provide atherapeutic dose in 10 ingestible formulations or fewer. In anotherexample, a therapeutic dose is provided in 20, 15, 10, 5 or 3 ingestibleformulations.

The compositions of the present invention may be in the form of adispersible dry powder for pulmonary delivery. Dry powder compositionsmay be prepared by processes known in the art, such as lyophilizationand jet milling, as disclosed in International Patent Publication No. WO91/16038 and as disclosed in U.S. Pat. No. 6,921,527, both of which areincorporated by reference. The composition of the present invention maybe placed within a suitable dosage receptacle in an amount sufficient toprovide a subject with a unit dosage treatment. The dosage receptaclemay be one that fits within a suitable inhalation device to allow forthe aerosolization of the dry powder composition by dispersion into agas stream to form an aerosol and then capturing the aerosol so producedin a chamber having a mouthpiece attached for subsequent inhalation by asubject in need of treatment. Such a dosage receptacle includes anycontainer enclosing the composition known in the art such as gelatin orplastic capsules with a removable portion that allows a stream of gas(e.g., air) to be directed into the container to disperse the dry powdercomposition. Such containers are exemplified by those shown in U.S. Pat.No. 4,227,522; U.S. Pat. No. 4,192,309; and U.S. Pat. No. 4,105,027.Suitable containers also include those used in conjunction with Glaxo'sVentolin® Rotohaler brand powder inhaler or Fison's Spinhaler® brandpowder inhaler. Another suitable unit-dose container which provides asuperior moisture barrier is formed from an aluminum foil plasticlaminate. The pharmaceutical-based powder is filled by weight or byvolume into the depression in the formable foil and hermetically sealedwith a covering foil-plastic laminate. Such a container for use with apowder inhalation device is described in U.S. Pat. No. 4,778,054 and isused with Glaxo's Diskhaler® (U.S. Pat. Nos. 4,627,432; 4,811,731; and5,035,237); all four of the US patents cited in this sentence areincorporated herein by reference.

The terms “transdermal delivery system”, “transdermal patch”, or “patch”refer to an adhesive system placed on the skin to deliver a timereleased dose of a drug(s) by passage from the dosage form through theskin to be available for distribution via the systemic circulation.Transdermal patches are a well-accepted technology used to deliver awide variety of pharmaceuticals, including, but not limited to,scopolamine for motion sickness, nitroglycerin for treatment of anginapectoris, clonidine for hypertension, estradiol for postmenopausalindications, and nicotine for smoking cessation. Patches suitable foruse in the present invention include, but are not limited to, (1) thematrix patch; (2) the reservoir patch; (3) the multi-laminatedrug-in-adhesive patch; and (4) the monolithic drug-in-adhesive patch;TRANSDERMAL AND TOPICAL DRUG DELIVERY SYSTEMS, pp. 249-297 (Tapash K.Ghosh et al. eds., 1997), hereby incorporated herein by reference. Thesepatches are well known in the art and generally available commercially.

In certain embodiments, the pharmaceutical composition features anysubject compound, and/or extract provided in an amount sufficient totreat Mild Cognitive Impairment, Alzheimer's disease, enhance long-termmemory, short-term memory, declarative memory, procedural memory orcognitive processes such as attention, executive function, reaction timeor learning in a patient by a statistically significant amount whenassessed by a standardized performance test.

In certain embodiments, the pharmaceutical composition features one ormore subject compound, and/or extract provided in an amount sufficientto enhance long-term memory in a patient by a statistically significantamount when assessed by one or more of a Rey Auditory and VerbalLearning Test (RAVLT), Cambridge Neuropsychological Test AutomatedBattery (CANTAB); a Children's Memory Scale (CMS); a Contextual MemoryTest; a Continuous Recognition Memory Test (CMRT); a DenmanNeuropsychology Memory Scale; a Fuld Object Memory Evaluation (FOME); aGraham-Kendall Memory for Designs Test; a Guild Memory Test; a Learningand Memory Battery (LAMB); a Memory Assessment Clinic Self-Rating Scale(MAC-S); a Memory Assessment Scales (MAS); a Randt Memory Test; aRecognition Memory Test (RMT); a Rivermead Behavioral Memory Test; aRussell's Version of the Wechsler Memory Scale (RWMS); a Test of Memoryand Learning (TOMAL); a Vermont Memory Scale (VMS); a Wechsler MemoryScale; and a Wide Range Assessment of Memory and Learning (WRAML);First-Last Name Association (Youngjohn J. R., et al., Archives ofClinical Neuropsychology 6:287-300 (1991)); Name-Face Association;Wechsler Memory Scale-Revised; (Wechsler, D., Wechsler MemoryScale-Revised Manual, NY, N.Y., The Psychological Corp. (1987));California Verbal Learning Test—Second Edition (Delis, D. C., et al.,The Californian Verbal Learning Test, Second Edition, Adult Version,Manual, San Antonio, Tex.: The Psychological Corporation (2000)); FacialRecognition (delayed non-matching to sample); Cognitive Drug Research(CDR) Computerized Assessment Battery-Wesnes; Buschke's SelectiveReminder Test (Buschke, H., et al., Neurology 24:1019-1025 (1974));Telephone Dialing Test; and Brief Visuospatial Memory Test-Revised.

Kits

This invention also provides kits for conveniently and effectivelyimplementing the methods of this invention. Such kits comprise anysubject compound, composition and/or extract, and a means forfacilitating compliance with methods of this invention. Such kitsprovide a convenient and effective means for assuring that the subjectto be treated takes the appropriate active in the correct dosage in thecorrect manner. The compliance means of such kits includes any meanswhich facilitates administering the actives according to a method ofthis invention. Such compliance means include instructions, packaging,and dispensing means, and combinations thereof. Kit components may bepackaged for either manual or partially or wholly automated practice ofthe foregoing methods. In other embodiments involving kits, thisinvention contemplates a kit including compositions of the presentinvention, and optionally instructions for their use.

For example, the present invention also provides for kits containing atleast one dose of a subject compound, composition and/or extract, andoften many doses, and other materials for a treatment regimen. Forexample, in one embodiment, a kit of the present invention containssufficient subject composition for from five to thirty days andoptionally equipment and supplies necessary to measure one or moreindices relevant to the treatment regiment. In another embodiment, kitsof the present invention contain all the materials and supplies,including subject compositions, for carrying out any methods of thepresent invention. In still another embodiment, kits of the presentinvention, as described above, additionally include instructions for theuse and administration of the subject compositions.

In one embodiment, the invention is a pharmaceutical kit comprising oneor more subject compound, composition and/or extract in an amountsufficient to enhance long-term memory in a patient, a pharmaceuticallyacceptable carrier, and instructions (written and/or pictorial)describing the use of the subject compound, composition and/or extractfor enhancing memory.

Dosage

The dosage of any compound, composition and/or extract of the presentinvention will vary depending on the symptoms, age and body weight ofthe patient, the nature and severity of the disorder to be treated, theroute of administration, and the form of the subject composition. Any ofthe subject formulations may be administered in a single dose or individed doses. Effective dosages for the compositions of the presentinvention may be readily determined by techniques known to those ofskill in the art or as taught herein. The dosage may be selected toassuage the disorder in a subject in such a way as to provide at leastpartial relief if not complete relief. The skilled artisan may identifythis amount as provided herein as well as by using other methods knownin the art.

A therapeutically effective amount (i.e., dose) of a compound of thepresent invention can be determined by methods known in the art. Thetherapeutically effective quantities will depend on the age and on thegeneral physiological condition of the patient, the route ofadministration and the pharmaceutical formulation used. In certainembodiments, the therapeutic doses may be between about 10 mg/day and10,000 mg/day, between about 100 mg/day and 2,500 mg/day, or between 250mg to about 1,000 mg/day. In other embodiments, other ranges may beused, including, for example, 50-250 mg/day, 250-500 mg/day, and 500-750mg/day. The amount of the compound required for prophylactic treatment,referred to as a prophylactically-effective dosage, is generally thesame as described for therapeutic treatment.

In certain embodiments, a therapeutic low dose of a compound orcomposition of the invention is administered. In certain embodiments,the therapeutically effective dose is between about 50 mg/day to about150 mg/day. In certain embodiments, the therapeutic dose is betweenabout 10 mg/day and about 20 mg/day. In certain embodiments, thetherapeutic dose is between about 20 mg/day and about 30 mg/day. Incertain embodiments, the therapeutic dose is between about 30 mg/day andabout 40 mg/day. In certain embodiments, the therapeutic dose is betweenabout 40 mg/day and about 50 mg/day. In certain embodiments, thetherapeutic dose is between about 50 mg/day and about 60 mg/day. Incertain embodiments, the therapeutic dose is between about 60 mg/day andabout 70 mg/day. In certain embodiments, the therapeutic dose is betweenabout 70 mg/day and about 80 mg/day. In certain embodiments, thetherapeutic dose is between about 80 mg/day and about 90 mg/day. Incertain embodiments, the therapeutic dose is between about 90 mg/day andabout 100 mg/day. In certain embodiments, the therapeutic dose isbetween about 100 mg/day and about 110 mg/day. In certain embodiments,the therapeutic dose is between about 110 mg/day and about 120 mg/day.In certain embodiments, the therapeutic dose is between about 120 mg/dayand about 130 mg/day. In certain embodiments, the therapeutic dose isbetween about 130 mg/day and about 140 mg/day. In certain embodiments,the therapeutic dose is between about 140 mg/day and about 150 mg/day.Administration may be once a day, twice a day, or more often, and may bedecreased during a maintenance phase of the treatment of a disease ordisorder, e.g., once every second or third day instead of every day ortwice a day. The dose and the administration frequency will depend onthe clinical signs, which confirm maintenance of the remission phase,with the reduction or absence of at least one or more clinical signs ofthe acute phase known to the person skilled in the art.

An effective dose or amount, and any possible affects on the timing ofadministration of the formulation, may need to be identified for anyparticular composition of the present invention. This may beaccomplished by routine experiment as described herein, using one ormore groups of animals, or in human trials if appropriate. Theeffectiveness of any subject composition and method of treating may beassessed by administering the composition and assessing the effect ofthe administration by measuring one or more applicable indices, andcomparing the post-treatment values of these indices to the values ofthe same indices prior to treatment.

The precise time of administration and amount of any particular subjectcomposition that will yield the most effective treatment in a givenpatient will depend upon the activity, pharmacokinetics, andbioavailability of a subject composition, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage and type of medication),route of administration, and the like.

While the subject is being treated, his or her health may be monitoredby measuring one or more of the relevant indices at predetermined timesduring the treatment period. Treatment, including composition, amounts,times of administration and formulation, may be optimized according tothe results of such monitoring. The patient may be periodicallyreevaluated to determine the extent of improvement by measuring the sameparameters. Adjustments to the amount(s) of subject compositionadministered and possibly to the time of administration may be madebased on these re-evaluations.

Treatment may be initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage may be increased bysmall increments until the optimum therapeutic effect is attained.

The use of the subject compositions may reduce the required dosage forany individual agent contained in the compositions because the onset andduration of effect of the different agents may be complimentary.

Toxicity and therapeutic efficacy of subject compositions may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ and the ED₅₀.

The data obtained from the cell culture assays and animal studies may beused in formulating a range of dosage for use in humans. The dosage ofany subject composition lies typically within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

Methods of Use

Certain aspects of the invention relate treating a subject having animpairment in memory and/or cognition. The subject can have animpairment in memory consolidation (the process of storing newinformation in long term memory), an impairment in short term memoryprocesses, an impairment in long-term memory, an impairment indeclarative memory or an impairment in procedural memory. The subjectsare treated with the compounds described herein to enhance, preventand/or restore long-term memory function and performance, e.g., toimprove the process of storing new information in long term memory inhumans (memory consolidation) or to improve short term memory.

One aspect of the invention relates to a method, comprising the step ofadministering to a subject an amount of a pomegranate extract; wherein

the extract contains an active fraction comprising at least onepolyphenol; and

the extract has been prepared by:

a juicing and manufacturing process, optionally followed byfractionalization by centrifugation and/or ultrafiltration, before beingdried;

a juicing and manufacturing process, optionally followed byfractionalization by centrifugation and/or ultrafiltration, before beingadsorbed into the polymeric chromatographic resin, washed with water,and eluted with a solvent; or

a juicing and manufacturing process, optionally followed byfractionalization by centrifugation and/or ultrafiltration, before beingadsorbed into the polymeric chromatographic resin, washed with water,eluted with a solvent, and subsequently dried.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract has been prepared by ajuicing and manufacturing process, optionally followed byfractionalization by centrifugation and/or ultrafiltration, before beingdried

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract has been prepared by ajuicing and manufacturing process, optionally followed byfractionalization by centrifugation and/or ultrafiltration, before beingadsorbed into the polymeric chromatographic resin, washed with water,and eluted with a solvent.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract has been prepared by ajuicing and manufacturing process, optionally followed byfractionalization by centrifugation and/or ultrafiltration, before beingadsorbed into the polymeric chromatographic resin, washed with water,eluted with a solvent, and subsequently dried.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is dried by spray drying.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is dried under vacuum (e.g.40° C. at 50 mbar).

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is fractionalized bycentrifugation.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is fractionalized byultrafiltration.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solvent is an alcohol (e.g.ethanol).

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the resin is a polyvinyl-styrene divinylbenzene resin. In certain embodiments, concentration on such resins canbe achieved via an adsorption in water at normal pressure or undervacuum, followed by extensive washing with water to remove highly polarconstituents like sugars, fibers, and minerals. Sugar elution during thewashing steps is followed with a standard refractometer and measured asBrix contant. Elution is then initiated by changing the buffer to morepolar solvent like ethanol, methanol or mix of ethanol in water. Inaddition, in some embodiments, whole molecule elution can be followed byUV detection at 205 nm for example. See, for example, the “Purificationof Ellagitannins” section of U.S. Pat. No. 7,638,640, which is herebyincorporated by reference for said section and in its entirety.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the resin is Amberlite XAD-16 (Rohm &Haas).

Another aspect of the invention relates to a method, comprising the stepof administering a compound of the invention to a subject.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is pure and isolated.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is administered as part ofan extract.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is adminstered as part ofan extract; and the extract is substantially free of polyphenols otherthan the compound.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is adminstered as part ofan extract; and the molar ratio of the moles of compound to the totalmoles of polyphenols in the extract is greater than about 0.99. Incertain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is adminstered as part ofan extract; and the molar ratio of the moles of compound to the totalmoles of polyphenols in the extract is greater than about 0.9. Incertain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is adminstered as part ofan extract; and the molar ratio of the moles of compound to the totalmoles of polyphenols in the extract is greater than about 0.85. Incertain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is adminstered as part ofan extract; and the molar ratio of the moles of compound to the totalmoles of polyphenols in the extract is greater than about 0.8. Incertain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is adminstered as part ofan extract; and the molar ratio of the moles of compound to the totalmoles of polyphenols in the extract is greater than about 0.75.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is whole fruit extract 1766.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is husk extract 1767.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is extract 31008.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is extract 31008-L or31008-H.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is extract 61109.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is extract 71109.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is extract 1767-1.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is extract 1767-2.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is extract 1767-3.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is extract 1767-4.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is extract 1767-5.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the extract is extract 1767-6.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is administered as part ofa nutraceutical composition.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is administered as part ofa functional food or functional nutrition product.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is administered as part ofa medical food or medical nutrition product.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is administered as part ofa dietary supplement.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is administered as part ofa pharmaceutical composition.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the subject is a human.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein said human is elderly.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein said human is not elderly.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein said human is less than 20 years old.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein said human is between 20 and 40 yearsold.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein said human is between 40 and 60 yearsold.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein said human is between 60 and 80 yearsold.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein said human is between 80 and 100 yearsold.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein said human is between 100 and 120 yearsold.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein said human is healthy.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein said human is not healthy.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the subject is a mammal.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the mammal is a veterinary animal.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the general cognition of the subject isimproved.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the memory of the subject is improved.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the general cognition of the subject ismaintained.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the memory of the subject is maintained.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein a neuron or plurality of neurons in saidsubject are protected.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the subject's neurons are partially orsubstantially protected.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the method treats or prevents aneurodegenerative disorder.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the rate of the progression of saidneurodegenerative disorder is decreased.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein onset of said neurodegenerative disorderis delayed.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the method treats or prevents metabolicsyndrome, type-II diabetes, dislipidemia, or obesity.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the method treats or prevents anamyloidosis-related condition in said subject.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the amyloidosis-related condition is MCIor AAMI.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the method treats or prevents ALS,Huntington's disease, Parkinson's disease, or Down syndrome.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the method treats or preventsAlzheimer's disease.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the method treats a memory impairment ina human.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the memory impairment results from oneor more of age-associated memory impairment, minimal cognitiveimpairment, amnesia, dementia, learning disabilities, memory impairmentassociated with toxicant exposure, brain injury, brain aneurysm,Parkinson's disease, head trauma, Huntington's disease, Pick's disease,Creutzfeldt-Jakob disease, stroke, schizophrenia, epilepsy, mentalretardation, Alzheimer's disease, age, age-associated memory impairment,Mild Cognitive Impairment, attention deficit disorder, attention deficithyperactivity disorder, Multiple Sclerosis, Anterior CommunicatingArtery Syndrome or AIDS-related dementia.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein memory is improved in the humanfollowing said administration.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the improvement in memory is animprovement in at least one measure selected from the group consistingof an improvement in short-term memory, long-term memory, memoryconsolidation, procedural memory and declarative memory.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein attention is improved in the humanfollowing said administration.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein executive function is improved in thehuman following said administration.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein reaction time is improved in the humanfollowing said administration.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein learning is improved in the humanfollowing said administration.

A person skilled in the art will be able to envision subjects that canbenefit from the methods described herein. For example, such subjectincludes peoples with suspected memory impairment of the Alzheimer'sdisease type, MCI, AAMI, Parkinson's disease, or ALS. Healthy elderlysubject showing sign of cognitive impairment may also benefit from thecompounds preventive affect of the present invention. One skilled in theart would recognize that the practitioner may apply different criteriafor a determination of signs memory impairment. Such criteria include,but are not limited to Diagnostic and Statistical Manual of MentalDisorders, third edition (DSM-III) Alzheimer's Disease Diagnostic andTreatment Center (ADDTC), International Statistical Classification ofDiseases, 10^(th) Revision (ICD-IO), National Institute of NeurologicalDisorders and Stroke-Association Internationale pour la Recherche etEnseignment en Neurosciences (NINDS-AIREN) and Diagnostic andStatistical Manual of Mental Disorders, Fourth Edition (DSM-IV). SeePohjasvaara et al, Stroke 2000, 31, 2952-2957. Clinical characterizationof a patient as mild cognitive impairment is well within the skill ofthe practitioner. Such testing of a patient to elucidate such acondition involves performing a series of mental tests. The methods forclinical diagnosis are widely reviewed and are discussed in, e.g.,Petersen et al, Arch. Neurol. 1999, 56, 303-308.

In addition, there is growing evidence that type-II Diabetes is a riskfactor for people to develop Alzheimer's disease; hence, people withpredisposition to type-II Diabetes may benefit from a preventive therapythat would slow down progression of neurodegenerative disorders. Ho, L.,W. Qin, et al. (2004). “Diet-induced insulin resistance promotesamyloidosis in a transgenic mouse model of Alzheimer's disease.” FASEB J18(7): 902-4; and Kojro, E. and R. Postina (2009). “RegulatedProteolysis of RAGE and AbetaPP as Possible Link Between Type 2 DiabetesMellitus and Alzheimer's Disease.” J Alzheimers Dis 16(4): 865-78.

Another aspect of the invention relates to a method of inhibitingaggregation of a peptide or protein, comprising the step of contactingthe peptide or protein with an effective amount of a compound of theinvention.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is a compound of theinvention, or pharmaceutically acceptable salt, biologically activemetabolite, solvate, hydrate, prodrug, enantiomer or stereoisomerthereof.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is pure and isolated.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the compound is in an extract.

Combination Therapy

In one aspect of the invention, a compound of the invention, or apharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof, or anextract or composition containing the same, can be used alone or incombination with another therapeutic agent to treat diseases. It shouldbe understood that the compounds of the invention can be used alone orin combination with an additional agent, e.g., a therapeutic agent, saidadditional agent being selected by the skilled artisan for its intendedpurpose. For example, the additional agent can be a therapeutic agentthat is art-recognized as being useful to treat the disease or conditionbeing treated by the compound of the present invention. The additionalagent also can be an agent that imparts a beneficial attribute to thetherapeutic composition e.g., an agent that affects the viscosity of thecomposition.

The combination therapy contemplated by the invention includes, forexample, administration of a compound of the invention, or apharmaceutically acceptable salt thereof, and additional agent(s) in asingle pharmaceutical formulation as well as administration of acompound of the invention, or a pharmaceutically acceptable saltthereof, and additional agent(s) in separate pharmaceuticalformulations. In other words, co-administration shall mean theadministration of at least two agents to a subject so as to provide thebeneficial effects of the combination of both agents. For example, theagents may be administered simultaneously or sequentially over a periodof time.

It should further be understood that the combinations included withinthe invention are those combinations useful for their intended purpose.The combination can also include more than one additional agent, e.g.,two or three additional agents if the combination is such that theformed composition can perform its intended function. In someembodiments, the additional agent could be another compound of theinvention, or a pharmaceutically acceptable salt, biologically activemetabolite, solvate, hydrate, prodrug, enantiomer or stereoisomerthereof.

In certain embodiments, the compounds, compositions and/or extracts ofthe present invention may be administered at least once per day incombination with a prescribed drug. For example, the composition of thepresent invention may be administered together with existinganticholinesterase drugs now prescribed for Alzheimer's, with variousanti-inflammatory agents, or with statins. In certain embodiments theprescribed drug is a cholinesterase inhibitor. In certain embodimentsthe prescribed drug is selected from the group consisting of Namenda®(memantine), Reminyl® (galantamine), Exelon® (rivastigmine), Aricept®(donepezil), Cognex® (tacrine), Carbex® (selegiline) and Eldepryl®(selegiline).

In another aspect, the composition of the present invention isadministered at least once per day in combination with a dietary ornutritional supplement believed to have beneficial health effects.Examples of dietary or nutritional supplements with which a compound,composition and/or extract may combined are below.

Coenzyme Q10 (also known as CoQ10, Q10, vitamin Q10, ubiquinone andubidecarenone), a benzoquinone compound synthesized naturally by thehuman body, is used by cells of the body in oxidative metabolism or cellrespiration and as an endogenous antioxidant. An “antioxidant” is asubstance that at least partially protects cells from free radicals,which are highly reactive chemicals often containing oxygen atoms, thatare capable of damaging important cellular components, such as DNA andlipids. The plasma level of CoQ10 has been used in studies as a measureof oxidative stress, a situation in which normal antioxidant levels arereduced. Various investigations have explored the usefulness of CoQ10 asa treatment for diseases, including, but not limited to, cancer andcardiovascular disease.

Idebenone, a synthetic analog of CoQ10, has been investigated in elderlypatients with dementia. Studies suggest that it may diminish nerve celldamage due to ischemia and facilitate memory and learning.

Huperzine A, a natural acetylcholinesterase inhibitor derived from theChinese herb Huperzia serrata, has antioxidant and neuroprotectiveproperties, and has been proposed as a disease-modifying treatment forAD.

Galantamine, an acetylcholinesterase inhibitor, is used to treatsymptoms of AD.

Vincamine and vinpocetine, a semisynthetic derivative of vincamine, analkaloid derived from the plant Vina minor L, are used in Europe, Japanand Mexico as pharmaceutical agents for the treatment of cerebrovascularand cognitive disorders.

Acetyl-L-carnitine, an acetylated derivative of carnitine, has beenshown to promote fatty acid beta-oxidation in liver and to prevent motornerve condition velocity slowing in diabetic rats.

Dehydroepiandrosterone (DHEA), a steroid, is being studied in theprevention of cancer. In the body, it is a precursor produced by theadrenal gland and converted to testosterone or the estrogens.

Phosphatidylcholine, a phospholipid that is a major component of cellmembranes, has putative activity as a cognition enhancer and incell-membrane repair

Gingko, an herb, has putative properties as a neuroprotective agent, anantioxidant, a free-radical scavenger, a membrane stabilizer, and aninhibitor of platelet-activating factor. Sherpina, V. S., et al.,American Family Physician 68(5) 923-926 (2003). Gingko extract also hasbeen shown to inhibit beta-amyloid deposition. Id.

Curcumin, an active ingredient in turmeric, which is in curry,purportedly has antiinflammatory and cholesterol-lowering properties.

Berberine, which is a quaternary ammonium salt from the group ofisoquinoline alkaloids. It is found in such plants as Berberis,goldenseal (Hydrastis canadensis), and Coptis chinensis, usually in theroots, rhizomes, stems, and bark. As a traditional medicine or dietarysupplement, berberine has showed some activity against fungalinfections, Candida albicans, yeast, parasites, and bacterial/viralinfections.

Ginseng, a Chinese herb, has been used for centuries in Asia as a curefor many maladies.

Research has shown that Vitamin E (DL-alpha-tocopherol), an essentialvitamin that functions as an antioxidant, can help preventcardiovascular disease and increase the immune response. It has beenhypothesized that Vitamin E and its analogs and derivatives may preventbrain cell damage by destroying toxic free radicals. The term “tocol”generally refers to 2-methyl-2-(4,8,12-trimethyltridecyl)chroman-6-ol;the term “tocopherol” generally refers to all mono, di, andtrimethyltocols, including, but not limited to, alpha-tocopherol(5,7,8-trimethyltocol), beta-tocopherol (5,8-dimethyltocol),gamma-tocopherol (7,8-dimethyltocol), delta-tocopherol (8-methyltocol),the term “tocotrienol” refers to2-methyl-2-(4,8,12-trimethyltrideca-3,7,11-trienyl)chroman-6-ol; and theterm “vitamin E” generally refers to all tocol and tocotrienolderivatives exhibiting qualitatively the biological activity ofalpha-tocopherol.

It is well-known that N-acetyl-cysteine (NAC) promotes cellularglutathione production, and thus reduces, or even prevents, oxidantmediated damage. Treatment with NAC provides beneficial effects in anumber of respiratory, cardiovascular, endocrine, infectious, and otherdisease settings.

B vitamins, such as folic acid, are known to reduce levels ofhomocysteine, an amino acid already linked, at high levels, to anincreased risk of heart attacks, strokes and Alzheimer's disease.

Lecithin, a lipid material composed of choline and inositol, is a majorcomponent of cell membranes. As used by producers of lecithin forcommercial use, the term “lecithin” refers to a complex mix ofphosphatides and other substances that contain phosphatidylcholine.

Choline (trimethyl ethanolamine), a quaternary saturated amineclassified as an essential nutrient by the Food and Nutrition Board ofthe Institute of Medicine, is a component of lecithin. Choline is neededby the body to make the neurotransmitter acetylcholine.

Omega-3 fatty acids are a family of unsaturated fatty acids that have incommon a final carbon-carbon double bond in the n-3 position; that is,the third bond from the methyl end of the fatty acid. Nutritionallyimportant n-3 fatty acids include α-linolenic acid (ALA),eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), all ofwhich are polyunsaturated. Some experts believe these compounds can helpregulate cholesterol in the body. They may also help protect the brainfrom cognitive problems associated with Alzheimer's disease.

Omega 6-fatty acids are a family of unsaturated fatty acids that have incommon a final carbon-carbon double bond in the n-6 position, that is,the sixth bond, counting from the end opposite the carboxyl group. Thebiological effects of the n-6 fatty acids are largely mediated by theirconversion to n-6 eicosanoids that bind to diverse receptors found inevery tissue of the body. The conversion of tissue arachidonic acid(20:4n-6) to n-6 prostaglandin and n-6 leukotriene hormones providesmany targets for pharmaceutical drug development and treatment todiminish excessive n-6 actions in atherosclerosis, asthma, arthritis,vascular disease, thrombosis, immune-inflammatory processes, and tumorproliferation. Competitive interactions with the n-3 fatty acids affectthe relative storage, mobilization, conversion and action of the n-3 andn-6 eicosanoid precursors.

Deprenyl (selegiline, Eldepryl®), a monoamine oxidase inhibitor, isprescribed for the treatment of early-stage Parkinson's disease andsenile dementia.

The formulations of the invention can be used alone or in combinationwith other pharmaceuticals or herbals to prolong mental health, tomaintain or enhance cognitive functioning or memory, or to preservemental or physical well-being and health. The formulations can also beused to prevent or treat effects of a number of ailments, including, butnot limited to, Alzheimer's disease; Parkinson's disease; heart disease;arthritis; age-related degeneration; functional impairments; diabetes;cancer; and other diseases having an impact on cognitive function.

The effectiveness of the compositions and methods of the presentinvention can be assayed by a variety of protocols. The effects ofincreasing cognitive function in a human subject can be determined bymethods routine to those skilled in the art including, but not limitedto, both paper and pencil, and computer tests. One of skill in the artcan also directly measure PP2A methylation levels, tau proteinphosphorylation levels, neurofibrillary tangle formation andneurodegeneration in animal models.

The combination therapy contemplated by the invention includes, forexample, administration of a compound of the invention and additionalagent(s) in a single formulation as well as administration of a compoundof the invention and additional agent(s) in separate formulations. Inother words, co-administration shall mean the administration of at leasttwo agents to a subject so as to provide the beneficial effects of thecombination of both agents. For example, the agents may be administeredsimultaneously or sequentially over a period of time.

It should further be understood that the combinations included withinthe invention are those combinations useful for their intended purpose.The combination can also include more than one additional agent, e.g.,two or three additional agents if the combination is such that theformed composition can perform its intended function.

Exemplary Tests for Cognitive Function

There are a variety of tests for cognitive function, especially learningand memory testing (see, for example, United States Patent ApplicationPublication No. 2010/0010097). Learning and/or memory tests include, forexample, Inhibitory Avoidance Test (also referred to herein as “PassiveAvoidance Test”), contextual fear conditioning, visual delay non-matchto sample, spatial delay non-match to sample, visual discrimination,Barnes circular maze, Morris water maze, radial arm maze tests, RayAuditory-Visual Learning Test, the Wechsler Logical Memory Test, and theProvidence Recognition Memory Test.

An exemplary Inhibitory Avoidance Test utilizes an apparatus thatconsists of a lit chamber that can be separated from a dark chamber by asliding door. At training, the animal is placed in the lit chamber forsome period of time, and the door is opened. The animal moves to thedark chamber after a short delay—the step-through latency—which isrecorded. Upon entry into the dark chamber, the door is shut closed anda foot shock is delivered. Retention of the experience is determinedafter various time intervals, e.g., 24 or 48 hours, by repeating thetest and recording the latency. The protocol is one of many variants ofthe passive avoidance procedures (for review, see Rush (1988) Behav.Neural. Biol. 50:255).

An exemplary maze testing embodiment is the water maze working memorytest. In general, the method utilizes an apparatus which consists of acircular water tank. The water in the tank is made cloudy by theaddition of milk powder. A clear plexiglass platform, supported by amovable stand rest on the bottom of the tank, is submerged just belowthe water surface. Normally, a swimming rat cannot perceive the locationof the platform but it may recall it from a previous experience andtraining, unless it suffers from some memory impairment. The time takento locate the platform is measured and referred to as the latency.During the experiment, all orientational cues such as ceiling lights,etc., remain unchanged. Longer latencies are generally observed withrats with some impairment to their memory.

Another memory test includes the eyeblink conditioning test, whichinvolves the administration of white noise or steady tone that precedesa mild air puff which stimulates the subject's eyeblink. With trainingthe auditory cue is sufficient to stimulate the eyeblinking response.This response is impaired in the presence of memory deficiencies.

Still another memory test which can be used is fear conditioning, e.g.,either “cued” and “contextual” fear conditioning. In one embodiment, afreeze monitor administers a sequence of stimuli (sounds, shock) andthen records a series of latencies measuring the recovery from shockinduced freezing of the animal.

Another memory test for the lesioned animals is a holeboard test, whichutilizes a rotating holeboard apparatus containing (four) open holesarranged in a 4-corner configuration in the floor of the test enclosure.A mouse is trained to poke its head into a hole and retrieve a foodreward from a “baited” hole which contains a reward on every trial.There is a food reward (e.g., Fruit Loops®) in every exposed hole whichis made inaccessible by being placed under a screen. The screen allowsthe odor of the reward to emanate from the hole, but does not allowaccess to the reinforcer. When an individual hole is baited, a reward isplaced on top of the screen, where it is accessible. The entireapparatus rests on a turntable so that it may be rotated easily toeliminate reliance on proximal (e.g., olfactory) cues. A start tube isplaced in the center of the apparatus. The subject is released from thetube and allowed to explore for the baited (“correct”) hole. Theperformance of the mouse in this assay is affected by memory impairment.

Another model for measuring memory impairment makes use offornix-lesioned animals for testing the ability of compounds to modulatememory consolidation, as well as for side effects and toxicity. Ingeneral, the subject method utilizes an animal which has beenmanipulated to create at least partial disruption of fornix-mediatedsignalling to the hippocampus, the disruption affecting memoryconsolidation and learned behavior in the animal. The animal isconditioned with a learning or memory regimen which results in learnedbehavior in the mammal in the absence of the fornix lesion. Compoundsare administered to the animal in order to assess their effects onmemory consolidation. An increase in learned behavior, relative to theabsence of the test agents, indicates that the administered combinationenhances memory consolidation.

Another memory test especially developed for use in pharmaceuticalstudies is the Providence Recognition Memory Test. This test consists ofone pictorial and one verbal assessment of long-term declarative memory.In each of the two modes, the patient views stimuli on a computer screenand is later asked to recognize those stimuli in a two-alternativeforced-choice format. The pictorial assessment mode consists of twoparts: a study phase and a recognition phase. In the study phase,patients view a series of 120 pictures, for 3 seconds each. They aretold to look at the pictures and remember them, so that they canrecognize them later. In the recognition phase, patients view picturestwo at a time and are asked to indicate by button press which of the twopictures they saw in a study phase. Recognition memory testing occurs atten minutes, one hour, and 24 hours after the end of the study phase.The verbal assessment mode consists of two parts: a study phase and arecognition phase. In the study phase, patients view a series of 60sentences one at a time. They are asked to read the sentences aloud andremember them, so that they can recognize them later. Each sentenceremains on the computer screen until the patient has finished reading italoud. If patients read words incorrectly, the examiner supplies thecorrect word or words. In the recognition phase, patients view sentencestwo at a time and are asked to indicate by button press which of the twosentences they saw in the study phase. Recognition memory testing occursat ten minutes, one hour, and 24 hours after the end of the study phase.

In the methods of the present invention, retention of the learnedbehavior can be determined, for example, after at least about 12-24hours, 14-22 hours, 16-20 hours and or 18-19 hours after completion ofthe learning phase to determine whether the agents promote memoryconsolidation. In a particular embodiment, retention of the learnedbehavior can be determined 24 hours after completion of the learningphase.

In addition to models for studying memory consolidation, models toassess side effects of amphetamine compounds on behavior have beenutilized including locomotor activity models. An exemplary locomotoractivity test utilizes an apparatus that consists of photocell activitycages with a grid of photocell beams placed around the cage. The animalsare placed in individual activity cages some period of time prior toadministration of agents. Locomotor activity is measured by the numberof interruptions of the photoelectric beam during a given period oftime.

As used herein, a “control mammal” can be an untreated lesion mammal(i.e., a lesion animal receiving no agents or not the same combinationsto be assessed), a trained control mammal (i.e., a mammal that undergoestraining to demonstrate a learned behavior without any lesion) and/or anuntrained control mammal (i.e., a mammal with or without a lesion, thatreceives no training to demonstrate a learned behavior).

DEFINITIONS

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

As used here, the term “anti-aggregation compound” refers to a compoundthat exhibits anti-aggregation properties, i.e., inhibits the formationof protein or peptide soluble or insoluble aggregate in vitro and/or invivo. For purpose of this definition, such effect can be quantified andtested in vitro as is known in the art, for example, as described in theexamples in the following references, each of which are herebyincorporated by reference. Ono, K., Y. Yoshiike, et al. (2003). “Potentanti-amyloidogenic and fibril-destabilizing effects of polyphenols invitro: implications for the prevention and therapeutics of Alzheimer'sdisease.” J Neurochem 87(1): 172-81; Riviere, C., T. Richard, et al.(2007). “Inhibitory activity of stilbenes on Alzheimer's beta-amyloidfibrils in vitro.” Bioorg Med Chem 15(2): 1160-7; and Riviere, C., T.Richard, et al. (2008). “New polyphenols active on beta-amyloidaggregation.” Bioorg Med Chem Lett 18(2): 828-31.

“Treating” is used herein to refer to any treatment of, or preventionof, or inhibition of a disorder or disease in a subject and includes byway of example: (a) preventing the disease or disorder from occurring ina subject that may be predisposed to the disease or disorder, but hasnot yet been diagnosed as having it; (b) inhibiting the disease ordisorder, i.e., arresting or delaying its progression; or (c) relievingor ameliorating the disease or disorder, i.e., causing regression.

The term “heteroatom” as used herein is art-recognized and refers to anatom of any element other than carbon or hydrogen. Illustrativeheteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur andselenium.

The term “alkyl” means an aliphatic or cyclic hydrocarbon radicalcontaining from 1 to 12 carbon atoms. Representative examples of alkylinclude, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl,neopentyl, n-hexyl, 2-methylcyclopentyl, and 1-cyclohexylethyl.

The term “alkylene” is art-recognized, and as used herein pertains to abidentate moiety obtained by removing two hydrogen atoms of an alkylgroup, as defined above.

The term “alkenyl” as used herein means a straight or branched chainhydrocarbon containing from 2 to 10 carbons and containing at least onecarbon-carbon double bond formed by the removal of two hydrogens.Representative examples of alkenyl include, but are not limited to,ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl,5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

The term “alkynyl” as used herein means a straight or branched chainhydrocarbon group containing from 2 to 10 carbon atoms and containing atleast one carbon-carbon triple bond. Representative examples of alkynylinclude, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl,3-butynyl, 2-pentynyl, and 1-butynyl.

The term “carbocyclyl” as used herein means monocyclic or multicyclic(e.g., bicyclic, tricyclic, etc.) hydrocarbons containing from 3 to 12carbon atoms that is completely saturated or has one or more unsaturatedbonds, and for the avoidance of doubt, the degree of unsaturation doesnot result in an aromatic ring system (e.g. phenyl). Examples ofcarbocyclyl groups include 1-cyclopropyl, 1-cyclobutyl, 2-cyclopentyl,1-cyclopentenyl, 3-cyclohexyl, 1-cyclohexenyl and 2-cyclopentenylmethyl.

The term “heterocyclyl”, as used herein include non-aromatic, ringsystems, including, but not limited to, monocyclic, bicyclic andtricyclic rings, which can be completely saturated or which can containone or more units of unsaturation, for the avoidance of doubt, thedegree of unsaturation does not result in an aromatic ring system, andhave 3 to 12 atoms including at least one heteroatom, such as nitrogen,oxygen, or sulfur. For purposes of exemplification, which should not beconstrued as limiting the scope of this invention, the following areexamples of heterocyclic rings: azepines, azetidinyl, morpholinyl,oxopiperidinyl, oxopyrrolidinyl, piperazinyl, piperidinyl, pyrrolidinyl,quinicludinyl, thiomorpholinyl, tetrahydropyranyl and tetrahydrofuranyl.The heterocyclyl groups of the invention are substituted with 0, 1, 2,3, 4 or 5 substituents independently selected from the group consistingof alkyl, alkenyl, alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy,alkoxy, alkenyloxy, alkynyloxy, carbocycyloxy, heterocycyloxy,haloalkoxy, fluoroalkyloxy, sulfhydryl, alkylthio, haloalkylthio,fluoroalkylthio, alkyenylthio, alkynylthio, sulfonic acid,alkylsulfonyl, haloalkylsulfonyl, fluororalkylsulfonyl, alkenylsulfonyl,alkynylsulfonyl, alkoxysulfonyl, haloalkoxysulfonyl,fluororalkoxysulfonyl, alkenyloxysulfonyl, alkynyloxysulfony,aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfinyl,fluororalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl,haloalkoxysulfinyl, fluororalkoxysulfinyl, alkenyloxysulfinyl,alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl,haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl,fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy,alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy,haloalkylsulfonyloxy, fluororalkylsulfonyloxy, alkenylsulfonyloxy,alkynylsulfonyloxy, haloalkoxysulfonyloxy, fluororalkoxysulfonyloxy,alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy,haloalkylsulfinyloxy, fluororalkylsulfinyloxy, alkenylsulfinyloxy,alkynylsulfinyloxy, alkoxysulfinyloxy, halo alkoxysulfinyloxy,fluororalkoxysulfinyloxy, alkenyloxysulfinyloxy, alkynyloxysulfinyloxy,aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, cyano,nitro, azido, phosphinyl, phosphoryl, silyl, silyloxy, and any of saidsubstituents bound to the heterocyclyl group through an alkylene moiety(e.g. methylene).

The term “aryl,” as used herein means a phenyl group, naphthyl oranthracenyl group. The aryl groups of the present invention can beoptionally substituted with 1, 2, 3, 4 or 5 substituents independentlyselected from the group consisting of alkyl, alkenyl, alkynyl, halo,haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkyenyloxy, alkynyloxy,carbocycyloxy, heterocycyloxy, haloalkoxy, fluoroalkyloxy, sulfhydryl,alkylthio, haloalkylthio, fluoroalkylthio, alkyenylthio, alkynylthio,sulfonic acid, alkylsulfonyl, haloalkylsulfonyl, fluororalkylsulfonyl,alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl, haloalkoxysulfonyl,fluororalkoxysulfonyl, alkenyloxysulfonyl, alkynyloxysulfony,aminosulfonyl, sulfinic acid, alkylsulfinyl, haloalkylsulfinyl,fluororalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, alkoxysulfinyl,haloalkoxysulfinyl, fluororalkoxysulfinyl, alkenyloxysulfinyl,alkynyloxysulfiny, aminosulfinyl, formyl, alkylcarbonyl,haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carboxy, alkoxycarbonyl, haloalkoxycarbonyl,fluoroalkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,alkylcarbonyloxy, haloalkylcarbonyloxy, fluoroalkylcarbonyloxy,alkenylcarbonyloxy, alkynylcarbonyloxy, alkylsulfonyloxy, haloalkylsulfonyloxy, fluororalkylsulfonyloxy, alkenylsulfonyloxy,alkynylsulfonyloxy, haloalkoxysulfonyloxy, fluororalkoxysulfonyloxy,alkenyloxysulfonyloxy, alkynyloxysulfonyloxy, alkylsulfinyloxy,haloalkylsulfinyloxy, fluororalkylsulfinyloxy, alkenylsulfinyloxy,alkynylsulfinyloxy, alkoxysulfinyloxy, haloalkoxysulfinyloxy,fluororalkoxysulfinyloxy, alkenyloxysulfinyloxy, alkynyloxysulfinyloxy,aminosulfinyloxy, amino, amido, aminosulfonyl, aminosulfinyl, cyano,nitro, azido, phosphinyl, phosphoryl, silyl, silyloxy, and any of saidsubstituents bound to the aryl group through a methylene, ethylene orpropylene moiety.

The term “arylene,” is art-recognized, and as used herein pertains to abidentate moiety obtained by removing two hydrogen atoms from adjacentcarbons of an aryl ring, as defined above.

The term “arylalkyl” or “aralkyl” as used herein means an aryl group, asdefined herein, appended to the parent molecular moiety through an alkylgroup, as defined herein. Representative examples of aralkyl include,but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, and2-naphth-2-ylethyl.

The term “heteroaryl” as used herein include aromatic ring systems,including, but not limited to, monocyclic, bicyclic and tricyclic rings,and have 3 to 12 atoms including at least one heteroatom, such asnitrogen, oxygen, or sulfur. For purposes of exemplification, whichshould not be construed as limiting the scope of this invention:azaindolyl, benzo(b)thienyl, benzimidazolyl, benzofuranyl, benzoxazolyl,benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzoxadiazolyl,furanyl, imidazolyl, imidazopyridinyl, indolyl, indolinyl, indazolyl,isoindolinyl, isoxazolyl, isothiazolyl, isoquinolinyl, oxadiazolyl,oxazolyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridinyl,pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl,pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl,thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl,thienyl, thiomorpholinyl, triazolyl or tropanyl. The heteroaryl groupsof the invention are substituted with 0, 1, 2, 3, 4 or 5 substituentsindependently selected from the group consisting of alkyl, alkenyl,alkynyl, halo, haloalkyl, fluoroalkyl, hydroxy, alkoxy, alkyenyloxy,alkynyloxy, carbocycyloxy, heterocycyloxy, haloalkoxy, fluoroalkyloxy,sulfhydryl, alkylthio, haloalkylthio, fluoroalkylthio, alkyenylthio,alkynylthio, sulfonic acid, alkylsulfonyl, haloalkylsulfonyl,fluororalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, alkoxysulfonyl,haloalkoxysulfonyl, fluororalkoxysulfonyl, alkenyloxysulfonyl,alkynyloxysulfony, aminosulfonyl, sulfinic acid, alkylsulfinyl,haloalkylsulfinyl, fluororalkylsulfinyl, alkenylsulfinyl,alkynylsulfinyl, alkoxysulfinyl, haloalkoxysulfinyl,fluororalkoxysulfinyl, alkenyloxysulfinyl, alkynyloxysulfiny,aminosulfinyl, formyl, alkylcarbonyl, haloalkylcarbonyl,fluoroalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy,alkoxycarbonyl, haloalkoxycarbonyl, fluoroalkoxycarbonyl,alkenyloxycarbonyl, alkynyloxycarbonyl, alkylcarbonyloxy,haloalkylcarbonyloxy, fluoroalkylcarbonyloxy, alkenylcarbonyloxy,alkynylcarbonyloxy, alkylsulfonyloxy, haloalkylsulfonyloxy,fluororalkylsulfonyloxy, alkenylsulfonyloxy, alkynylsulfonyloxy,haloalkoxysulfonyloxy, fluororalkoxysulfonyloxy, alkenyloxysulfonyloxy,alkynyloxysulfonyloxy, alkylsulfinyloxy, haloalkylsulfinyloxy,fluororalkylsulfinyloxy, alkenylsulfinyloxy, alkynylsulfinyloxy,alkoxysulfinyloxy, haloalkoxysulfinyloxy, fluororalkoxysulfinyloxy,alkenyloxysulfinyloxy, alkynyloxysulfinyloxy, aminosulfinyloxy, amino,amido, aminosulfonyl, aminosulfinyl, cyano, nitro, azido, phosphinyl,phosphoryl, silyl, silyloxy, and any of said substituents bound to theheteroaryl group through an alkylene moiety (e.g. methylene).

The term “heteroarylene,” is art-recognized, and as used herein pertainsto a bidentate moiety obtained by removing two hydrogen atoms fromadjacent atoms of a heteroaryl ring, as defined above.

The term “heteroarylalkyl” or “heteroaralkyl” as used herein means aheteroaryl, as defined herein, appended to the parent molecular moietythrough an alkyl group, as defined herein. Representative examples ofheteroarylalkyl include, but are not limited to, pyridin-3-ylmethyl and2-(thien-2-yl)ethyl.

The term “halo” or “halogen” means —Cl, —Br, —I or —F.

The term “haloalkyl” means an alkyl group, as defined herein, wherein atleast one hydrogen is replaced with a halogen, as defined herein.Representative examples of haloalkyl include, but are not limited to,chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and2-chloro-3-fluoropentyl.

The term “fluoroalkyl” means an alkyl group, as defined herein, whereinall the hydrogens are replaced with fluorines.

The term “hydroxy” as used herein means an —OH group.

The term “alkoxy” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Representative examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, andhexyloxy. The terms “alkyenyloxy”, “alkynyloxy”, “carbocycyloxy”, and“heterocycyloxy” are likewise defined.

The term “haloalkoxy” as used herein means an alkoxy group, as definedherein, wherein at least one hydrogen is replaced with a halogen, asdefined herein. Representative examples of haloalkoxy include, but arenot limited to, chloromethoxy, 2-fluoroethoxy, trifluoromethoxy, andpentafluoroethoxy. The term “fluoroalkyloxy” is likewise defined.

The term “aryloxy” as used herein means an aryl group, as definedherein, appended to the parent molecular moiety through an oxygen. Theterm “heteroaryloxy” as used herein means a heteroaryl group, as definedherein, appended to the parent molecular moiety through an oxygen. Theterms “heteroaryloxy” is likewise defined.

The term “arylalkoxy” or “arylalkyloxy” as used herein means anarylalkyl group, as defined herein, appended to the parent molecularmoiety through an oxygen. The term “heteroarylalkoxy” is likewisedefined. Representative examples of aryloxy and heteroarylalkoxyinclude, but are not limited to, 2-chlorophenylmethoxy,3-trifluoromethyl-phenylethoxy, and 2,3-dimethylpyridinylmethoxy.

The term “sulfhydryl” or “thio” as used herein means a —SH group.

The term “alkylthio” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through a sulfur.Representative examples of alkylthio include, but are not limited,methylthio, ethylthio, tert-butylthio, and hexylthio. The terms“haloalkylthio”, “fluoroalkylthio”, “alkyenylthio”, “alkynylthio”,“carbocycylthio”, and “heterocycylthio” are likewise defined.

The term “arylthio” as used herein means an aryl group, as definedherein, appended to the parent molecular moiety through an sulfur. Theterm “heteroarylthio” is likewise defined.

The term “arylalkylthio” or “aralkylthio” as used herein means anarylalkyl group, as defined herein, appended to the parent molecularmoiety through an sulfur. The term “heteroarylalkylthio” is likewisedefined.

The term “sulfonyl” as used herein refers to —S(═O)₂— group.

The term “sulfonic acid” as used herein refers to —S(═O)₂OH.

The term “alkylsulfonyl” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through a sulfonylgroup, as defined herein. Representative examples of alkylsulfonylinclude, but are not limited to, methylsulfonyl and ethylsulfonyl. Theterms “haloalkylsulfonyl”, “fluororalkylsulfonyl”, “alkenylsulfonyl”,“alkynylsulfonyl”, “carbocycylsulfonyl”, “heterocycylsulfonyl”,“arylsulfonyl”, “aralkylsulfonyl”, “heteroarylsulfonyl” and“heteroaralkylsulfonyl” are likewise defined.

The term “alkoxysulfonyl” as used herein means an alkoxy group, asdefined herein, appended to the parent molecular moiety through asulfonyl group, as defined herein. Representative examples ofalkoxysulfonyl include, but are not limited to, methoxysulfonyl,ethoxysulfonyl and propoxysulfonyl. The terms “haloalkoxysulfonyl”,“fluororalkoxysulfonyl”, “alkenyloxysulfonyl”, “alkynyloxysulfonyl”,“carbocycyloxysulfonyl”, “heterocycyloxysulfonyl”, “aryloxysulfonyl”,“aralkyloxysulfonyl”, “heteroaryloxysulfonyl” and“heteroaralkyloxysulfonyl” are likewise defined.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The term “aminosulfonyl” as used herein means an amino group, as definedherein, appended to the parent molecular moiety through a sulfonylgroup.

The term “sulfinyl” as used herein refers to —S(═O)— group. Sulfinylgroups are as defined above for sulfonyl groups. The term “sulfinicacid” as used herein refers to —S(═O)OH.

The term “oxy” refers to a —O— group.

The term “carbonyl” as used herein means a —C(═O)— group.

The term “thiocarbonyl” as used herein means a —C(═S)— group.

The term “formyl” as used herein means a —C(═O)H group.

The term “alkylcarbonyl” as used herein means an alkyl group, as definedherein, appended to the parent molecular moiety through a carbonylgroup, as defined herein. Representative examples of alkylcarbonylinclude, but are not limited to, acetyl, 1-oxopropyl,2,2-dimethyl-1-oxopropyl, 1-oxobutyl, and 1-oxopentyl. The terms“haloalkylcarbonyl”, “fluoroalkylcarbonyl”, “alkenylcarbonyl”,“alkynylcarbonyl”, “carbocycylcarbonyl”, “heterocycylcarbonyl”,“arylcarbonyl”, “aralkylcarbonyl”, “heteroarylcarbonyl”, and“heteroaralkylcarbonyl” are likewise defined.

The term “carboxy” as used herein means a —CO₂H group.

The term “alkoxycarbonyl” as used herein means an alkoxy group, asdefined herein, appended to the parent molecular moiety through acarbonyl group, as defined herein. Representative examples ofalkoxycarbonyl include, but are not limited to, methoxycarbonyl,ethoxycarbonyl, and tert-butoxycarbonyl. The terms “haloalkoxycarbonyl”,“fluoroalkoxycarbonyl”, “alkenyloxycarbonyl”, “alkynyloxycarbonyl”,“carbocycyloxycarbonyl”, “heterocycyloxycarbonyl”, “aryloxycarbonyl”,“aralkyloxycarbonyl”, “heteroaryloxycarbonyl”, and“heteroaralkyloxycarbonyl” are likewise defined.

The term “alkylcarbonyloxy” as used herein means an alkylcarbonyl group,as defined herein, appended to the parent molecular moiety through anoxygen atom. Representative examples of alkylcarbonyloxy include, butare not limited to, acetyloxy, ethylcarbonyloxy, andtert-butylcarbonyloxy. The terms “haloalkylcarbonyloxy”,“fluoroalkylcarbonyloxy”, “alkenylcarbonyloxy”, “alkynylcarbonyloxy”,“carbocycylcarbonyloxy”, “heterocycylcarbonyloxy”, “arylcarbonyloxy”,“aralkylcarbonyloxy”, “heteroarylcarbonyloxy”, and“heteroaralkylcarbonyloxy” are likewise defined.

The term “alkylsulfonyloxy” as used herein means an alkylsulfonyl group,as defined herein, appended to the parent molecular moiety through anoxygen atom. The terms “haloalkylsulfonyloxy”,“fluororalkylsulfonyloxy”, “alkenylsulfonyloxy”, “alkynylsulfonyloxy”,“carbocycylsulfonyloxy”, “heterocycylsulfonyloxy”, “arylsulfonyloxy”,“aralkylsulfonyloxy”, “heteroarylsulfonyloxy”,“heteroaralkylsulfonyloxy”, “haloalkoxysulfonyloxy”,“fluororalkoxysulfonyloxy”, “alkenyloxysulfonyloxy”,“alkynyloxysulfonyloxy”, “carbocycyloxysulfonyloxy”,“heterocycyloxysulfonyloxy”, “aryloxysulfonyloxy”,“aralkyloxysulfonyloxy”, “heteroaryloxysulfonyloxy” and“heteroaralkyloxysulfonyloxy” are examples of thereof.

The term “amino” as used herein refers to —NH₂ and substitutedderivatives thereof wherein one or both of the hydrogens areindependently replaced with substituents selected from the groupconsisting of alkyl, haloalkyl, fluoroalkyl, alkenyl, alkynyl,carbocycyl, heterocycyl, aryl, aralkyl, heteroaryl, heteroaralkyl,alkylcarbonyl, haloalkylcarbonyl, fluoroalkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, carbocycylcarbonyl, heterocycylcarbonyl, arylcarbonyl,aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl and thesulfonyl and sulfinyl groups defined above; or when both hydrogenstogether are replaced with an alkylene group (to form a ring whichcontains the nitrogen). Representative examples include, but are notlimited to methylamino, acetylamino, and dimethylamino.

The term “amido” as used herein means an amino group, as defined herein,appended to the parent molecular moiety through a carbonyl.

The term “cyano” as used herein means a —C≡N group.

The term “nitro” as used herein means a —NO₂ group.

The term “azido” as used herein means a —N₃ group.

The term “phosphinyl” as used herein includes —PH₃ and substitutedderivatives thereof wherein one, two or three of the hydrogens areindependently replaced with substituents selected from the groupconsisting of alkyl, haloalkyl, fluoroalkyl, alkenyl, alkynyl,carbocycyl, heterocycyl, aryl, aralkyl, heteroaryl, heteroaralkyl,alkoxy, haloalkoxy, fluoroalkyloxy, alkenyloxy, alkynyloxy,carbocycyloxy, heterocycyloxy, aryloxy, aralkyloxy, heteroaryloxy,heteroaralkyloxy, and amino.

The term “phosphoryl” as used herein refers to —P(═O)OH₂ and substitutedderivatives thereof wherein one or both of the hydroxyls areindependently replaced with substituents selected from the groupconsisting of alkyl, haloalkyl, fluoroalkyl, alkenyl, alkynyl,carbocycyl, heterocycyl, aryl, aralkyl, heteroaryl, heteroaralkyl,alkoxy, haloalkoxy, fluoroalkyloxy, alkenyloxy, alkynyloxy,carbocycyloxy, heterocycyloxy, aryloxy, aralkyloxy, heteroaryloxy,heteroaralkyloxy, and amino.

The term “silyl” as used herein includes H₃Si— and substitutedderivatives thereof wherein one, two or three of the hydrogens areindependently replaced with substitutents selected from alkyl,haloalkyl, fluoroalkyl, alkenyl, alkynyl, carbocycyl, heterocycyl, aryl,aralkyl, heteroaryl, and heteroaralkyl. Representative examples includetrimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS),tert-butyldimethylsilyl (TBS/TBDMS), triisopropylsilyl (TIPS), and[2-(trimethylsilyl)ethoxy]methyl (SEM).

The term “silyloxy” as used herein means a silyl group, as definedherein, is appended to the parent molecule through an oxygen atom.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations.

As used herein, a “carbohydrate” (or, equivalently, a “sugar”) is asaccharide (including monosaccharides, oligosaccharides andpolysaccharides) and/or a molecule (including oligomers or polymers)derived from one or more monosaccharides, e.g., by reduction of carbonylgroups, by oxidation of one or more terminal groups to carboxylic acids,by replacement of one or more hydroxy group(s) by a hydrogen atom, anamino group, a thiol group or similar heteroatomic groups, etc. The term“carbohydrate” also includes derivatives of these compounds. In somecases, the carbohydrate may be a pentose (i.e., having 5 carbons) or ahexose (i.e., having 6 carbons); and in certain instances, thecarbohydrate may be an oligosaccharide comprising pentose and/or hexoseunits, e.g., including those described above.

“Carbohydrate” and “sugar” as used herein also includes sugar-mimeticsand sugar-like moieties. Sugar-mimetics are well known to one ofordinary skill in the art and include those described in detail in“Essentials of Glycobiology” Edited by Varki, A., et al, Cold SpringHarbor Laboratory Press. Cold Spring Harbor, N.Y. 2002. For example,sugar-mimetic groups contemplated by the present invention includecyclitols, such as a cycloalkane containing one hydroxyl group on eachof three or more ring atoms, as defined by IUPAC convention. In otherembodiments, such cyclitol moieties include inositols such asscyllo-inositol. Suitable sugar-like moieties include acyclic sugargroups. Such groups include linear alkylols and erythritols, to name buta few. It will be appreciated that sugar groups can exist in eithercyclic or acyclic form. Accordingly, acyclic forms of a sugar group arecontemplated by the present invention as a suitable sugar-like moieties.

The term “gallic acid equivalents” is a well-known term of art,referring to the use of gallic acid as a standard for quantification ofthe phenol content of various analytes by the Folin-Ciocalteau assay.

The term “extract” or “botanical extract” as used herein refers to aproduct prepared by separating, by chemical or physical process,medicinally active portions of a plan from the inactive or inertcomponents.

As used herein, the term “cognitive function” refers to the ability toperform mental tasks, such as thinking, learning, judging, remembering,computing, controlling motor functions, and the like. The expression“resilience of cognitive function” refers to the ability of functionalelements of cognitive function to resist deterioration over time.

As used herein, the terms “therapeutically effective amount,”“memory-enhancing amount”, and “cognition enhancing amount” are usedinterchangeably to refer to the amount of the composition of theinvention that results in a therapeutic or beneficial effect, includinga subject's perception of health or general well-being, following itsadministration to a subject.

In particular, “cognitive function enhancing amount” refers to thatamount of the composition of the present invention that will noticeablyimpact the ability to perform mental tasks, as measured by tests formemory, computation, attention, or other mental or cognitive attribute,or as suggested by an individual's perception of his or her abilities inthese realms.

The terms “dietary supplement” and “nutritional supplement” are usedinterchangeably herein to mean (1) a product intended to supplement thediet that bears or contains one or more of the following dietaryingredients: (A) a vitamin, (B) a mineral, (C) an herb or otherbotanical, (D) an amino acid, (E) a dietary substance for use by man tosupplement the diet by increasing the total dietary intake; and/or (F) aconcentrate, metabolite, constituent, extract, or combination of anyingredient described in clause (A), (B), (C), (D), or (E); and (2) aproduct that (A) (i) is intended for ingestion; (B) is not representedfor use as a conventional food or as a sole item of a meal or the diet;and (C) is labeled as a dietary supplement.

The term “food” as used herein refers to (1) articles used for food ordrink for man or other animals; (2) chewing gum; and (3) articles usedfor components of any such article. The term “functional food” or“functional nutrition product” refers to a food or nutrition productthat is sold (e.g. in a supermarket or online) without any restrictions.The term “medical food” or “medical nutrition product” refers to a foodor nutrition product with is prescribed by a physician. Foods ornutrition products may be solids, liquids, gels, powders or gases.Examples of solids are fruit-based drinks, coffee-based drinks,tea-based drinks, sport drinks, nutrition bars, snack foods, gums,cereals, candies, baby formulas, energy drinks, adult nutritionaldrinks, health drinks, and other food products. The term “sports drink”refers to a beverage that is supposed to rehydrate athletes, as well asrestoring electrolytes, sugar and other nutrients, for example,Gatorade, POWERade, and AU Sport. As used herein, the term “energydrink” refers to a beverage, including, but not limited to, Jolt Cola,Red Bull and similar products, that contains legal stimulants, vitaminsand minerals; these products are formulated to give the user a burst ofenergy. The term “adult nutritional drink” as used herein refers to suchproducts as Ensure, Longetics® or a similar product. The term “healthdrink” refers to any beverage purported to have beneficial healtheffects, including, but not limited to, reducing inflammation;supporting the immune system; neutralizing infectious agents; preventingclogged arteries, preserving cognitive function and inhibiting cancergrowth.

EXEMPLIFICATION

The invention will be more readily understood by reference to thefollowing examples, which are included merely for purposes ofillustration of certain aspects and embodiments of the presentinvention, and are not intended to limit the invention.

Example 1 Screening Assay for Pomegranate Compounds which InhibitAggregation of Aβ(25-35)

Monomeric Aβ(25-35) purchased from Bachem Pharma, was dissolved indistilled sterile water at 4° C., then sonicated for about 1 minute. Thepeptide stock solution was then aliquoted and stored at −20° C. Allsteps were carried out at 4° C. to prevent Aβ(25-35) polymerization.

Fractions of polyphenols were all standardized to 10 mg/mL, before beingassessed and stored at −20° C. Measurement of inhibition was performedby following the kinetic of Aβ(25-35) polymerization in a reactionmixture containing 70 μL phosphate buffer, 10 μL Thioflavin-T (100 μMstock solution), 10 μL MeOH (10 mM final concentration) pH 7.2 and 10 μLof Aβ(25-35) (100 μM final concentration) adapting known in the artprocedures. LeVine, H., 3rd (1993). “Thioflavine T interaction withsynthetic Alzheimer's disease beta-amyloid peptides: detection ofamyloid aggregation in solution.” Protein Sci. 2(3): 404-10. WhenAβ(25-35) was added to the buffer solution, the solution was sonicatedfor an additional minute.

To study the inhibitory activity of the fractions, the extracts werediluted to the final concentration in MeOH and 10 μL was added to themixture at 4° C. Fluorescence spectroscopy was recorded with theexcitation at 450 nm and emission at 490 nm. The polymerization kineticswas monitored at 25° C. between 0 and 120 minutes. The IC₅₀ wascalculated by using a least-square fitting technique to match theexperimental data with a sigmoidal curve. The IC₅₀ was the effectiveconcentration dose of the compound inhibiting the formation of Aβfibrils to 50% of the control value.

Pomegranate (Punica granatum, Wonderful variety) was extracted usingthree different buffers to yield fractions enriched with molecules ofincreased polarity. The pomegranate was blended and extracted withwater, butanol, and ethyl-acetate buffers. The three fractions weretested in the assay as described in Example 1 at 10 mg/mL each. Theresults showed that the ethyl acetate fraction contained the highestactivity, being able to inhibit over 65% of the aggregation (FIG. 11).

The ethyl-acetate, butanol and water fraction were furthersub-fractionated by centrifugal partition chromatography and purecompounds were isolated using standard HPLC methods. The molecules wereidentified using HPLC/MS and NMR studies. Each molecule was used atpurity above 90% in the assay for aggregation described in Example 1.The results are depicted in FIGS. 12-18 and summarized below.

Fraction A3J inhibited aggregation with an IC₅₀ of 10 μM and NMRconfirmed the identity of the molecule to be Punicalin; fraction A1Dinhibited aggregation with an IC₅₀ of 11 μM. MS data confirmed theidentity of the molecule to be Pedunculagin; fraction W12 inhibitedaggregation with an IC₅₀ of 6 μM and was confirmed to be Punicalagin;and fraction A3I inhibited aggregation with an IC₅₀ of 9.3 μM. Finally,fraction A3G inhibited aggregation with an IC₅₀ of 6.8 μM and NMR and MSdata confirmed the identity of the molecule to be Tellimagrandin.Corilagin inhibited aggregation with an IC₅₀ of 65 μM.

Example 2 Measuring Inhibition of Aβ-Oligomer Induced Neuronal CellDeath

The fractions that were able to inhibits oligomer formation were alsotested for their ability to inhibit Aβ-oligomer induced neuronal celldeath using P12 cells, a well known and accepted model of neuronal cellculture.

Beta Amyloid peptide (Aβ) 25-35 trifluoroacetate salt (ref. H-1192, lot2000718) was purchased from Bachem (Bubebdorf, Switzerland). ThiazolylBlue Tetrazolium Bromide (MTT, ref M2128) was purchased fromSigma-Aldrich (Saint-Quentin Fallvier, France). Albumin Bovine FractionV (BSA, ref 160069) was purchased from MPBio (Irvine, USA).

Cells: Rat Pheochromocytoma cells (PC12) were maintained in DMEM (4.5g/L glucose) supplemented with 10% Horse Serum (HS), 5% Fetal BovineSerum (FCS) and 100 U/mL penicillin and 100 μg/mL Streptomycin at 37° C.under 5% CO₂.

Preparation of Aβ (25-35): It has been reported that Aβ oligomers aremore toxic to neurons than monomers. Therefore, the Aβ (25-35) that wasused for this study was pre-aggregated prior to use. Aβ was dissolve to1 mM in water and stored at −20° C. Aβ (25-35) was incubated in 37° C.for 3 days to induce aggregation just prior to use.

Determination of hydro-alcoholic Pomegranate extracts ability to protectPC12 cells against Aβ insult: The ability of Pomegranate extracts toprotect PC12 cells from Aβ (25-35) oligomer induced toxicity wasdetermined by measuring reduction of MTT to MTT formazan, which reflectscell viability. PC12 cells were plated in 96 wells culture platesandonce confluent, were pretreated with different concentrations ofPomegranate extracts (0.1% DMSO) for 1 h in DMEM 0.1% BSA. The cellswere then incubated with or without pre-aggregated Aβ (25-35) at 10 μMfor an additional 24 h. After the 24 h, MTT solution 300 μg/mL in cellculture medium (stock solution 3 mg/mL in PBS) was added for 2 h at 37°C. The medium was then removed and the formazan crystals are dissolvedin 100 μL of DMSO. The level of MTT reduction and thus cell viabilitywas determined by a colorimetric assay measuring the optical density at595 nm using a microplate reader (BioTek EL808).

As shown in FIG. 24, Extracts 1766 and 1767 were able to protect PC12neuronal cells from the toxicity of Aβ (25-35) oligomers. Extract 1766is a hydro-alcoholic extract for the whole pomegranate fruit, Extract1767 is a hydro-alcoholic extract of the pomegranate husk and For theextraction, whole fruit and husks were separated out and cut, crushedand placed into a blender. The resulting pieces were incubated in ahydro-alcoholic solution (EtOH/water), 80/20) and submitted to agitationat room temperature for 4 hours. After additional maceration in a lightprotected environment, the solutions were filtered and dried undervacuum (40° C. at 50 mbar) and then stored at −20° C. until use offurther fractionation. Subfractions of Extract 1767 were made and testedfor their ability to protect against Aβ-inducted cytotoxicity in PC12cells. Fractions 1767-2 and 1767-3 were shown to be the most active.Further analysis, concentration and testing of these two fractions ledto the identification of punicalagin as the natural compound responsiblefor the observed activity. DMSO was used as a carrier and showed nointerference on the assay.

Example 4 Extraction Procedure

The pomegranate extract 31008 containing the specific molecules wasprepared using an extraction procedure, based on adsorption ofpolyphenols in a standard polymer adsorption-based column as described.The pomegranates (e.g., Mollar variety) were juiced using a standardjuicing and manufacturing process and clarified by centrifugation beforebeing adsorbed into the polymeric chromatographic resin as pure juice.The resin Amberlite XAD-16 (Rohm & Haas) was packed into semipreparative columns and loaded with the extracted juice. The column waswashed with water to remove the sugars until completion (Brix levelswere below 0.1%). The polyphenols were eluted with 100% ethanol. Theremaining ethanol was evaporated under vacuum to produce a concentratedextract containing 4.5 g of total polyphenol per liter as determinedusing the Folin assay for total polyphenol content.

Extract 31008 was shown to contain the molecules Punicalagins,Punicalin, Tellimagrandin, and Pedunculagin, using HPLC-MSidentification for the compounds described above.

This technique is a modification of methods known in the art asdescribed by several published method for purification of polyphenolsform various plant and berry. Tuck, K. L. and P. J. Hayball (2002).“Major phenolic compounds in olive oil: metabolism and health effects.”J Nutr Biochem 13(11): 636-644; and Schieber, A., P. Hilt, et al.(2003). “A new process for the combined recovery of pectin and phenoliccompounds from apple pomace.” InnoVations Food Sci. Emerging Technol. 4:99-107.

Example 4 Animal Testing—Morris Water Maze

The extract 31008 was tested in an animal model of Alzheimer's diseaseexpressing both the Amyloid mutant London mutations and the prenisilin-1human mutation. Animals in this model develop plaques by 4 months of ageand memory deficits by 6 months. Dense plaque load is visible after 7months.

In one set of experiments, four-month old APP-PS1 transgenic mice werefed with a fixed dose of approximately 97 mg total polyphenols/kg/daysof the extract via their drinking water. After 3 months of feeding, themice (then 7 months old) were tested in the Morris-Water mazed spatialtest.

The Morris Water Maze was performed during days 84-87 of treatment. Thepool (a white, circular vessel 1 m in diameter) contained water at 20°C. with titanium-dioxide as an odorless, nontoxic additive to hide theescape platform (1 cm beneath the water level). Swimming of each mousewas videotaped and analyzed (Ethovision, Noldus information Technology,Wageningen, the Netherlands). Prior to training, each mouse was placedon top of the platform for 15 seconds. For place navigation tests, micewere trained to locate the hidden platform in five blocks of threetrials over three consecutive days. Each trial consists of a forced swimtest of maximum 120 seconds, followed by 60 seconds of rest. The timeeach mouse needed to locate the platform was measured during the fiveconsecutive blocks of training to determine a learning curve for eachmouse.

24 hours after the final training, each animal underwent a probe trialwith the platform removed. Mice were allowed to search for the missingplatform for 60 seconds and the search time spent in each quadrant ofthe pool, as well as the number of crossings of the original platformposition was measured. As shown in FIG. 24, the mice fed with Extract31008 showed an increase performance in the probe test as demonstratedby the increased frequency of crossings of the area were the platformwas formally located.

Example 5 Animal Testing—Amyloid Plaque Load

As the level of amyloid plaque load has been shown to correlate with theprogress of amyloid based diseases, the effects of treatment with theextract 31008 was examined. Mice were sacrificed and their brainscollected and prepared for immune histochemistry using standard methods.Sagital vibratome sections (40 μm) were cut for free floatingincubations and stored at 4° C. until staining in PBS with 0.1% sodiumazide. Thirty consecutive sections per brain in the region of thesubiculum were selected for staining (laterale between 2.18 and 1.08mm). Sections 1, 7, 13, 19, 25 were stained with IHC for Aβ(anti-amyloid). Sections of all animals used were randomized forstaining and blind quantification.

Free floating sections were incubated in Nett-wells to stain allsections in one single assay and minimize intensity variation. Sectionswere washed twice in PBS and incubated for 15 minutes in hydrogenperoxide 1.5% in PBS and methanol (1:1) to remove endogenous peroxidaseactivity. After washing the sections three times in PBS containing 0.1%Triton X100 (PBST), the sections were blocked for 30 minutes in 10%Fetal Calf Serum (FCS) in PBST followed by an overnight incubation withthe biotinylated primary antibody in PBST with 10% FCS (anti-amyloidagainst N-terminal end of Aβ, labeled with biotin, using a dilution of1:2800). After rinsing, the sections were incubated in 0.01% trypsin inPBS for 15 minutes at 37° C., followed by an incubation withavidin-biotin peroxidase complex (Vectastain Elite ABC, Vector,Burlingame, Calif.). The signal was developed with 3,3′-diaminobenzidinetetrahydrochloride tablets (DAB, ICN, 1 tablet/10 mL Tris-HCl with 3 μLH₂O₂ per 10 mL). Sections were counterstained with Mayers hematoxylin,dehydrated in five steps (50, 70, 95 and 2×100%) in ethanol and xylene(Merck Eurolab) and mounted in Depex (Depex mounting medium, BDHLaboratory).

As shown in FIG. 25, the mice fed with the extract has a significantreduction in brain amyloid plaque load consistent with the effect of theidentified molecules on aggregation.

In a second set of experiments, two-months-old APP-PS1 transgenic micewere fed with a fixed dose of approximately 90 mg (Extract 31008-L-) and800 mg (Extract 31008-H—) total polyphenols/kg/days of a highly enrichedPunicalagin extract, Extract 31008, via their drinking water. Anothergroup was also fed with a highly enriched Punicalagin extract, Extract61109 at a dose of 468 mg/kg/d. A final group was fed a pomegranate huskderived extract 71109 at a dose of 120 mg/kg/d. After 4 months offeeding, the mice (then about 6.5 months old) were tested in theMorris-Water Maze spatial memory test.

As shown in FIG. 26, the mice fed with the extract 31008 at a dose of 97mg/day showed an increase performance during the probe test, as shown bythe increase in the time spent in the platform zone. On the other hand,mice fed the extract 31008 at a dose of 828 mg/day showed no change inperformance as compared to the control transgenic administered theisocaloric vehicle. This result illustrates a dose effect on theimprovement of memory with the employed pomegranate extract. Micereceiving extracts 61109 and 71109 showed a significantly improvedperformance in the probe test (P<0.05 with Tukey's Multiple ComparisonTest versus vehicle hAPP-Tg).

Example 6 Animal Testing—Aged Rats

The effect of oral administration of polyphenols from pomegranateextracts on the cognitive performance of aged rats was studied. Thismodel system was selected to evaluate the effects of the treatment inanimals displaying impaired brain function as a result of the ageingprocess in order to highlight potential benefits that may not bedetectable in normal young adults. Treatments were initiated in maleSprague-Dawley rats at the age of 19 months, corresponding approximatelyto the onset of cognitive impairments (see Martinez-Serrano A, BjörklundA. “Ex vivo nerve growth factor gene transfer to the basal forebrain inpresymptomatic middle-aged rats prevents the development of cholinergicneuron atrophy and cognitive impairment during aging” Proc Natl Acad SciUSA. 1998 Feb. 17; 95(4):1858-63; and Bisson J F, Nejdi A, Rozan P,Hidalgo S, Lalonde R, Messaoudi M. “Effects of long-term administrationof a cocoa polyphenolic extract (Acticoa powder) on cognitiveperformances in aged rats” Br J Nutr. 2008 July; 100(1):94-101, Epub2008 Jan. 8). Animal performance was mainly monitored using behavioraltests for swim task learning and social recognition memory.

Polyphenols were administered orally, by dilution in the drinking water,to reproduce the ultimately targeted mode of delivery. Experimentscompared two doses of polyphenol-enriched extracts from pomegranate.

a) Social Recognition. In the social recognition discrimination task,each aged rat was placed in its home cage together with a juvenile maleSprague-Dawley rat (<5 weeks old) for 5 minutes. Thirty minutes later,the same exact procedure was repeated with the same juvenile todetermine a second time the degree of interaction between the twoanimals. In animals with a proper functioning memory, less contact isexpected as the two animals have had previous interactions. Thirtyminutes after the second exposure, a novel juvenile rat was placed for 5minutes together with the aged rat, in order to measure whether theanimal could discriminate between the two different juvenileindividuals. During each period of interaction between the two animals,the total time of contact was measured to assess social recognition.This test was performed after 2.5 to 3 months of treatment.

As shown in FIG. 27, in the aged rats fed the low dose of extract 31008(15 mg gallic acid equivalent/kg/day) the decrease observed in the timespent with juvenile #1 during second exposure indicated improvedperformance in recognition memory as a result of treatment. Whensubsequently exposed to the new juvenile rat #2, the interaction timeincreased in aged rats treated with extract at 15 mg gallic acidequivalent/kg/day, showing that the treated aged rats can distinguishbetween the two different juvenile rats and have a functioning memory.On the other hand, isocaloric control-treated aged rats have comparableinteraction times for the 1^(st) and 2^(nd) exposure to juvenile rat #1,indicating an impairment in their recognition memory. Control aged ratsexposed to juvenile #2 show a comparable interaction time with all priorexposures to juvenile #1, indicating the control animals are unable todistinguish between these interactions, a hallmark of reduced cognitionand a decline in memory. (N=12 for isocaloric controls, N=14 for theextract-treated group). Aged rats treated with the extract at a dose of75 mg gallic acid equivalent/kg/day (N=10), unlike rats treated with th15 mg GAE, have a performance similar to the isocaloric control group,when exposed a second time to juvenile rat #1 or to a novel juvenile rat#2. Thus demonstrating the importance of dosing in this effect.

b) Morris Water Maze: Reversal Test. Aged rats were treated continuouslywith the extract 31008 (15 mg gallic acid equivalent/kg/day) or anisocaloric control for a period of 3 months starting at the age of 19months. Aged rats were tested for their performance in the reversaltask: following training and probe test, the platform was placed in theopposite quadrant (WEST→EAST) and the animal was subjected to threetraining sessions. In this test, the rapidity to re-adapt to a newplatform location in a similar task further evaluates learning abilityof the animals.

As shown in FIG. 28, aged rats treated with the extract at 15 mg gallicacid equivalent/kg/day were significantly more efficient at localizingthe platform in the reversal test, as compared to the isocaloric controlaged rats (one-way ANOVA, P<0.02; isocaloric control N=11; extract:N=14). This illustrates the beneficial effects of a low dose of extract(15 mg gallic acid equivalent/kg/day) on memory improvement andprotection versus the natural memory decline observed in control,non-treated aged rats. The observed effects of the 15 mg gallic acidequivalent/kg/day dose is consistent with the observations in the socialrecognition memory evaluation and the importance of proper dosing toachieve memory improvements.

Incorporation by Reference

All of the U.S. patents and U.S. patent application publications citedherein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by this disclosure.

1. A method comprising the step of administering to a subject an amountof Punicalin, Punicalagin, Pedunculagin, Tellimagrandin, Corilagin,Granatine A, Granatine B, Terminalin, Gallagyldilactone, Compound A or acompound of Formula I:

or pharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof; wherein,independently for each occurrence, X is

and R¹, R² and R³ are each independently hydrogen, alkyl, aralkyl,alkylcarboxy, or a sugar; or R¹ is hydrogen, alkyl, aralkyl,alkylcarboxy,

or a sugar, R² is hydrogen or

and R³ is

or R¹ is hydrogen, alkyl, aralkyl, alkylcarboxy, or a sugar, and R² andR³ taken together are


2. The method of claim 1, wherein the compound of the invention is pureand isolated.
 3. The method of claim 1, wherein the compound isadministered as part of a nutraceutical composition, pharmaceuticalcomposition, functional food, functional nutrition product, medicalfood, medical nutrition product, dietary supplement or botanical drug.4. The method of claim 1, wherein the subject is a human.
 5. The methodof claim 1, wherein the general cognition of the subject is improved. 6.The method of claim 1, wherein the method treats or prevents a cognitivedisorder, a neurodegenerative disorder, a metabolic syndrome, type-IIdiabetes, dislipidemia, or obesity.
 7. The method of claim 1, whereinthe method treats or prevents an amyloidosis-related condition in saidsubject.
 8. The method of claim 1, wherein the method treats or preventsALS, Huntington's disease, Parkinson's disease, or Down syndrome.
 9. Themethod of claim 1, wherein the method treats or prevents Alzheimer'sdisease,
 10. The method of claim 1, wherein the method treats acognitive impairment in a human.
 11. The method of claim 10, whereinmemory is improved in the human following said administration.
 12. Themethod of claim 10, wherein the improvement in memory is an improvementin at least one measure selected from the group consisting of animprovement in short-term memory, long-term memory, memoryconsolidation, procedural memory and declarative memory.
 13. The methodof claim 10, wherein attention is improved in the human following saidadministration.
 14. The method of claim 10, wherein reaction time isimproved in the human following said administration.
 15. The method ofclaim 10, wherein learning is improved in the human following saidadministration.
 16. A method comprising the step of administering to asubject an amount of a pomegranate extract; wherein the extract containsan active fraction comprising at least one polyphenol; and the extracthas been prepared by: a juicing and manufacturing process, optionallyfollowed by fractionalization by centrifugation and/or ultrafiltration,before being dried; a juicing and manufacturing process, optionallyfollowed by fractionalization by centrifugation and/or ultrafiltration,before being adsorbed into the polymeric chromatographic resin, washedwith water, and eluted with a solvent; or a juicing and manufacturingprocess, optionally followed by fractionalization by centrifugationand/or ultrafiltration, before being adsorbed into the polymericchromatographic resin, washed with water, eluted with a solvent, andsubsequently dried.
 17. A method of inhibiting aggregation of a peptideor protein, comprising the step of contacting the peptide or proteinwith an effective amount of Punicalin, Punicalagin, Pedunculagin,Tellimagrandin, Corilagin, Granatine A, Granatine B, Terminalin,Gallagyldilactone, Compound A or a compound of Formula I:

or pharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof; wherein,independently for each occurrence, X is

and R¹, R² and R³ are each independently hydrogen, alkyl, aralkyl,alkylcarboxy, or a sugar; or R¹ is hydrogen, alkyl, aralkyl,alkylcarboxy,

or a sugar, R² is hydrogen or

and R³ is

or R¹ is hydrogen, alkyl, aralkyl, alkylcarboxy, or a sugar, and R² andR³ taken together are


18. A pure and isolated compound, or a pharmaceutically acceptable salt,biologically active metabolite, solvate, hydrate, prodrug, enantiomer orstereoisomer thereof; wherein the compound is represented by formula I:

wherein independently for each occurrence X is

and R¹, R² and R³ are each independently hydrogen, alkyl, aralkyl,alkylcarboxy, or a sugar; or R¹ is hydrogen, alkyl, aralkyl,alkylcarboxy,

or a sugar, R² is hydrogen or

and R³ is

or R¹ is hydrogen, alkyl, aralkyl, alkylcarboxy, or a sugar, and R² andR³ taken together are


19. The compound of claim 18, wherein the compound is Compound A, or apharmaceutically acceptable salt, biologically active metabolite,solvate, hydrate, prodrug, enantiomer or stereoisomer thereof.